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Biotechnology Toward Medicinal Plants (MPs)

April 2024

April 2024

DOI:10.1007/978-981-97-1003-4_10

In book: Industrial Crop Plants (pp.255-292)

Authors:

Marina Silalahi

Universitas Kristen Indonesia

Anisa Anggraeni

University of Indonesia

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The use of medicinal plants (MPs) that produce a secondary metabolite continues to grow along with population growth. As a result, overexploitation occurs and causes species extinction and genetic erosion. This article aims to answer the question about the role of biotechnology in increasing the production of secondary metabolite and their conservation. The biotechnologies that can be used to increase secondary metabolite are tissue culture and bioreactor. Secondary metabolites of tissue culture MPs improved through methods such as such as (1) selection of high-yield lines; (2) optimization of culture conditions (nutrient media composition, inoculum density, temperature, light, agitation, and aeration); (3) elicitation; (4) addition of precursors; and (5) immobilization. Meanwhile, the conservation of MPs can be carried out by utilizing in vitro technology, especially in vitro cold storage and cryopreservation. This shows that humans, biotechnology, and plants have a close relationship and can be optimized to meet the MPs needs and their conservation. Nevertheless, the social impact of the use of biotechnology still needs to be studied.

Biosynthetic pathway of certain classes of plant secondary metabolites from glucose within plant cells (Isah et al. 2018)

…

Structures of some bioactive compounds used in the pharmaceutical industry (Wawrosch and Zotchev 2021; Chandra and Chandra 2011; Isah et al. 2018; Shinyuy et al. 2023)

…

The table below presents publications related to the utilization of bioreactors in the production of secondary metabolites from medicinal plants in Indonesia over the last 5 years

…

Figures - uploaded by Marina Silalahi

Content may be subject to copyright.

Content uploaded by Marina Silalahi

Content may be subject to copyright.

255© The Author(s), under exclusive license to Springer Nature Singapore Pte

Ltd. 2024

N. Kumar (ed.), Industrial Crop Plants, Interdisciplinary Biotechnological

Advances, https://doi.org/10.1007/978-981-97-1003-4_10

Biotechnology Toward Medicinal Plants

(MPs)

MarinaSilalahi, I.GustiAyuRaiSawitri, A.Z.Wakhidah, AnisaAnggraeni,

andEisyaH.Hidayati

Abstract The use of medicinal plants (MPs) that produce a secondary metabolite

continues to grow along with population growth. As a result, overexploitation occurs

and causes species extinction and genetic erosion. This article aims to answer the

question about the role of biotechnology in increasing the production of secondary

metabolite and their conservation. The biotechnologies that can be used to increase

secondary metabolite are tissue culture and bioreactor. Secondary metabolites of

tissue culture MPs improved through methods such as such as (1) selection of high-

yield lines; (2) optimization of culture conditions (nutrient media composition,

inoculum density, temperature, light, agitation, and aeration); (3) elicitation; (4)

addition of precursors; and (5) immobilization. Meanwhile, the conservation of

MPs can be carried out by utilizing invitro technology, especially invitro cold stor-

age and cryopreservation. This shows that humans, biotechnology, and plants have

a close relationship and can be optimized to meet the MPs needs and their conserva-

tion. Nevertheless, the social impact of the use of biotechnology still needs to be

studied.

Keywords Biotechnology · Medicinal plants · Tissue culture · Cryopreservation

M. Silalahi (*)

Faculty of Education and Teacher Training, Department of Biology Education, Universitas

Kristen Indonesia, East Jakarta, Indonesia

e-mail: marina.silalahi@uki.ac.id

I. Gusti Ayu Rai Sawitri · A. Anggraeni

The Ethnobiological Society of Indonesia, Cirebon, West Java, Indonesia

A. Z. Wakhidah

Biology Education Study Program, Faculty of Tarbiyah and Education, Islamic State Institute

Metro, Metro, Lampung, Indonesia

E. H. Hidayati

Faculty of Mathematics and Natural Sciences, Department of Biology, Universitas Indonesia,

Kota Depok, West Java, Indonesia

256

1 Introduction

Plants that produce various types of secondary metabolites such as alkaloids, avo-

noids, terpenoids, and steroids (Roy etal. 2022; Mohaddab etal. 2022; Isah etal.

2018; Guerriero etal. 2018) and have been used as ingredients for medicines are

known as medicinal plants (MPs). The MPs are often indistinguishable from aro-

matic plants, because almost all aromatic plants are used as medicinal ingredients

(Silalahi etal. 2023). The use of plants as traditional/modern medicine is more com-

mon than the use of plants for other purposes such as avoring, fragrances, insecti-

cides, dyes, and medicine. Around 80% of people depend on traditional MPs for

their livelihoods (Kayser 2018; Pant 2014), and in developing countries, almost

90% of people rely on these plants (El Sheikha 2017). Some MPs used as therapy

and have been described in modern medicine include Dioscorea deltoidea, Papaver

somniferum, Atropa belladonna, Rauvola serpentina, Hyoscyamus niger, Digitalis

lanata, Datura metel, Digitalis purpurea, Pilocarpusa bonandi, and Cinchona

ledriana (Alamgir and Alamgir 2017).

The use of MPs began 5000years ago and continues to grow along with popula-

tion growth (Pant 2014; Alamgir and Alamgir 2017). This is because MPs are con-

sidered to have low side effects (Sarwat etal. 2012). About a quarter of prescribed

medications contain plant extracts or active ingredients obtained from plant materi-

als (Kayser 2018). A total of 60% of anticancer (El Sheikha 2017; Wilson and

Roberts 2012) and 75% of infectious disease medicine are produced from natural or

analog products, although there are many challenges in MPs production (Wilson and

Roberts 2012). On the other hand, plant secondary metabolites/bioactive compounds

with therapeutic effects have very complex structures and make them difcult to

synthesize (Staniek etal. 2014; Dziggel etal. 2017; Fazili etal. 2022; Wawrosch and

Zotchev 2021). Conventional production of secondary metabolites/bioactive com-

pounds of MPs on a commercial scale suffers from several barriers due to (1) limited

supply/sources; (2) relatively small yields (<1% of plant dry weight); (3) natural

harvesting is often impractical; (4) harvesting is limited by seasonality; and (5) rela-

tively slow plant growth (Wilson and Roberts 2012); misidentication, genetic and

phenotypic variability, extract variability and instability, toxic components, and con-

taminants (Alamgir and Alamgir 2017). As a result, overexploitation of MPs occurs

and causes species extinction and genetic erosion (Pant 2014; Canter etal. 2005).

Biotechnology such as tissue culture, bioreactors, and cryopreservation should be

considered to increase yield and modify the potential of MPs (Reed etal. 2011). The

plant tissue culture has been proven to be a potential alternative for the production

of secondary metabolite/bioactive compounds, producing plant material (Pant 2014;

Bhojwani etal. 2013), and for the conservation of endangered, critical, and rare spe-

cies (Pant 2014; Tripathi and Tripathi 2003). In bioreactors, the production of sec-

ondary metabolite/bioactive compounds also provides stability and consistency

(Udomsin etal. 2020; Fidan etal. 2022; Carlo etal. 2021; Agisimanto etal. 2022),

which are challenging to achieve with conventional plant culture methods (Udomsin

etal. 2020). Meanwhile, cryopreservation is a long-term conservation method in

liquid nitrogen and provides an opportunity for the conservation of endangered MPs

(Tripathi and Tripathi 2003). In view of the future potential of MPs, this article aims

M. Silalahi et al.

257

to answer the question about the role of biotechnology in increasing the production

of secondary metabolite/bioactive compound and their conservation.

2 Medicinal Plants (MPs) andTheir Bioactive Compounds

Plants produce various primary metabolites (for growth and development) and sec-

ondary metabolites (for self-defense and environmental adaptation) (Raj and

Saudagar 2019; Wilson and Roberts 2012; Guerriero etal. 2018). Secondary metab-

olites are often considered as biochemical “side pathways” in metabolism as shown

in Fig.1 (Bernhoft 2010; Isah etal. 2018). In plants, secondary metabolites serve

various functions: (1) avonoids protect against free radicals generated during pho-

tosynthesis; (2) terpenoids attract pollinators or seed dispersers, or inhibit compet-

ing plants; and (3) alkaloids deter herbivores or insect attacks (phytoalexins)

(Bernhoft 2010). The production of secondary metabolite compounds (phytochem-

istry) signicantly depends on plant species, geographic location, climate condi-

tions, and soil factors (Raj and Saudagar 2019).

Compounds such as alkaloids, avonoids, terpenes, and steroids are biosynthe-

sized as plant defense strategies in response to disturbances in natural environmen-

tal conditions, which humans utilize as medicinal ingredients (Isah etal. 2018). For

example, avonoids exhibit therapeutic activities as antibacterial, antiviral, antioxi-

dant, anti-inammatory, antimutagenic, and anticarcinogenic agents (Roy et al.

2022). Taxol is a rare alkaloid diterpene complex with potent anticancer activity

(Cusido etal. 2014).

Fig. 1 Biosynthetic pathway of certain classes of plant secondary metabolites from glucose within

plant cells (Isah etal. 2018)

Biotechnology Toward Medicinal Plants (MPs)

258

Based on their processing, bioactive compounds derived from plant secondary

metabolites, which are used by humans as medicines, are categorized into two

groups: (1) used directly as drugs and (2) as raw materials for semisynthetic modi-

cations (Wawrosch and Zotchev 2021). In the pharmaceutical industry, specic

bioactive compound contents are often used as determinants of the quality of MPs

(Hussein and El-Anssary 2019). Figure2 presents commercially utilized plant sec-

ondary metabolites for addressing various severe diseases.

Fig. 2 Structures of some bioactive compounds used in the pharmaceutical industry (Wawrosch

and Zotchev 2021; Chandra and Chandra 2011; Isah etal. 2018; Shinyuy etal. 2023)

M. Silalahi et al.

259

3 Plant Tissue Culture

Medicinal plants can be cultivated in the following ways: (a) agricultural practices

at the eld level and (b) production of secondary metabolites/bioactive compounds

invitro (Alamgir and Alamgir 2017). Biotechnologies that are often used to pro-

duce secondary metabolites for the pharmaceutical industry include cell culture,

tissue culture, genetic transformation (Cardoso etal. 2019; Siahsar etal. 2011), and

adventitious root culture (Baque etal. 2012; Siahsar etal. 2011). Tissue culture is

invitro method that has an important role in increasing the production of secondary

metabolites/bioactive compounds (Vanisree et al. 2004; Alamgir and Alamgir

2017; Mohanlall 2020) and was introduced in the late 1960s as a possible tool to

study and produce plant secondary metabolites (Vanisree etal. 2004; Bourgaud

etal. 2001). The tissue culture of MPs refers to the growth and propagation of cells,

tissues, and organs in an aseptic media and controlled environment (George and

Manuel 2013). Several bioactive compounds of high economic value have been

successfully produced through invitro techniques including diosgenin-derived ste-

roid hormone precursors, digitalis glycosides, berberine isoquinoline alkaloids,

Taxol, and paclitaxel (Alamgir and Alamgir 2017). Therefore, it can be used to

meet the demand for plant-based medicines in large quantities (Sharma etal. 2010;

George and Manuel 2013; Mukhopadhyay 2023), commercial (Mukhopadhyay

2023), high quality (Tripathi and Tripathi 2003), and sustainable (Mukhopadhyay

2023). Table 1 shows various bioactive compounds produced through in vitro

culture.

Some of the advantages of producing secondary metabolites/bioactive com-

pounds through invitro culture include:

1. in vitro production of phytochemicals can continue throughout the year

like a plant

2. it does not depend on climatic and soil conditions

3. it is free of microbes and insects (Bhojwani etal. 2013; Cardoso etal. 2019;

Alamgir and Alamgir 2017)

4. it provides an opportunity to increase the yield of natural compounds (Bhojwani

etal. 2013; Pant 2014)

5. it can induce cells to synthesize novel compounds by manipulating growth con-

ditions (Bhojwani etal. 2013; Gandhi etal. 2015)

6. predictable/stable continuous production

7. easier extraction and purication (Wawrosch and Zotchev 2021; Motolinía-

Alcántara etal. 2021)

8. reduced content of toxic compounds

9. generating secondary metabolism in MPs can be altered in innovative ways

(Gandhi etal. 2015)

10. genetic transformation to change the biosynthetic pathway of target metabolites

(Canter etal. 2005).

Increasing secondary metabolites/bioactive compounds through tissue culture

can be conducted with the use of biotic or abiotic elicitors (Alamgir and Alamgir

2017). Single-cell and meristem cultures can be effectively used to eradicate

Biotechnology Toward Medicinal Plants (MPs)

260

Table 1 Tissue culture method for the production of secondary metabolites of medicinal

plants (MPs)

No. Scientic name Secondary metabolites

Medium with

growth

regulator (GH)

Type tissue

culture References

1Bacopa

monnieri

L.Pennell

Bacosides MS medium

with BAP,

2,4-D, kinetin,

IAA, TDZ

Cell

suspension

cultures and

organ

cultures

Fazili etal.

(2022);

Sudheer etal.

(2023)

2Bidens pilosa

Chlorogenic acids MS medium

with 2,4-D and

BAP

Kalus Ramabulana

etal. (2021)

3Catharanthus

roseus (L.)

G.Don.

Ajmalicine

(monoterpene indole

alkaloid)

Zenk medium

with NAA and

BAP

Cell culture Esyanti and

Muspiah

(2006)

4Curcuma longa

Curcuminoids Nutrient broth

with

acetosyringone

Hairy root

cultures

Sandhya and

Archana (2023)

5Fagonia indica

(Schweinf.)

Hadidi

Caffeic acid, rutin,

hydroxybenzoic acid

MS medium Callus Begum etal.

(2021)

6Hybanthus

enneaspermus

Domin

L-3,4-

dihydroxyphenylalanine

MS medium

with NAA

Root culture Saeed etal.

(2017)

7Melia

azedarach L.

Rutin, quercetin, and

kaempferol

MS medium

NAA, BAP,

Kinetin

Cell culture Ahmadpoor

etal. (2023)

8Ocimum

basilicum L.

Flavonoids, phenolic

acids

MS medium

with GA3

Cell

suspension

culture

Açıkgöz (2020)

9Ocimum

sanctum L.

Eugenol, phenol,

avonoid

Medium MA

with Phe and

MeJA

Shoots

cultures

Autaijamsripon

etal. (2023)

10 Panax ginseng

(Burkill)

H.L.Li

Ginsenoside Medium MS

with IBA

Cell

suspension

culture

Hao etal.

(2020)

11 Persicaria

minor (Huds.)

Opiz

Sesquiterpenes Medium MS

2,4-D and

NAA

Cell

suspension

culture

Sellapan etal.

(2018)

12 Primula veris

subsp. veris L.

Primulic acid II MS medium

with NAA and

Kinetin

Adventitious

root cultures

Sarropoulou

etal. (2023)

13 Ruta

graveolens L.

Furanocoumarins,

furoquinoline alkaloids,

catechin, phenolic acids

Medium

Linsmaier and

Skoog with

NAA and BAP

Shoot

cultures

Szewczyk etal.

(2023)

(continued)

M. Silalahi et al.

261

Table 1 (continued)

No. Scientic name Secondary metabolites

Medium with

growth

regulator (GH)

Type tissue

culture References

14 Sageretia thea

(Osbeck)

M.C.Johnst.

Phenolics MS medium

with 2,4-D,

NAA, Kinetin

Cell

suspension

cultures

Kim etal.

(2023)

15 Salvia

fruticosa Mill.

Oleanolic acid, ursolic

acid

Linsmaier and

Skoog medium

with 2,4 D

Cell

suspension

culture

Kümmritz etal.

(2016)

16 Scutellaria

bornmuelleri

Hausskn. ex

Bornm.

Chrysin, wogonin,

baicalein

MS liquid

medium with

IBA

Hairy root

culture

Gharari etal.

(2020)

17 Silybum

marianum

L.Gaertn.

Silymarin MS medium

with NAA,

BAP, coconut

water

Callus Ebrahimzadeh

etal. (2023)

18 Stelechocarpus

burahol

(Blume)

Hook.f. &

Thomson

Flavonoid MS medium

with picloram,

2,4-D

Callus Habibah etal.

(2016)

19 Swertia

chirayita

(Roxb.)

H.Karst.

Amarogentin and

mangiferin

MS medium

with IBA and

kinetin

Shoot

cultures

Gupta and

Sood (2023)

20 Withania

somnifera (L.)

Withaferin Medium MS Hairy root

culture

Thilip etal.

(2019)

pathogens from planting material and thereby dramatically increase the yield of

stabilized cultivars (Brown and Thorpe 1995).

4 Increasing theNumber ofSecondary Metabolites/Bioactive

Compounds Through Tissue Culture

There are many advantages to the production of secondary metabolites/bioactive

compounds through tissue culture as discussed previously, but some of the com-

pounds that have been produced on a commercial scale are often below expectations

(Bhojwani etal. 2013). Researchers have developed various methods to increase the

secondary metabolites through tissue culture such as (1) selection of high-yield

lines, (2) optimization of culture conditions (nutrient media composition, inoculum

density, temperature, light, agitation, and aeration), (3) elicitation, (4) addition of

precursors, and (5) immobilization (Wawrosch and Zotchev 2021; Murthy etal.

2014). Constraints in the production of natural ingredients from conventional

Biotechnology Toward Medicinal Plants (MPs)

262

cultivation include relatively low yields, slow growth, and high variations in product

accumulation, making invitro culture technology the preferred choice for second-

ary metabolite production (Singh and Kaur 2014).

4.1 Selection ofHigh-Yield Lines

Selecting cell lines that produce high biomass and metabolites is critical in optimiz-

ing cell productivity using invitro culture (Isah et al. 2018). The expression of

synthetic pathways for the mass production of secondary metabolites can be

improved by selecting the right cell lines (Dörnenburg and Knorr 1995; Raj and

Saudagar 2019). Genetic transformation methods using Agrobacterium tumefaciens

(Siahsar etal. 2011), recombinant DNA (Bourgaud etal. 2001), and genome “edit-

ing” (Niazian 2019) are able to produce “special” MPs with different secondary

metabolite proles and high yields. Verpoorte et al. (2002) stated that screening,

selection, and optimization of media can increase secondary metabolites of MPs up

to 20–30 times (Verpoorte etal. 2002). Recombinant DNA technology will make it

possible to directly modify the expression of genes related to biosynthesis and engi-

neer secondary metabolites (Bourgaud etal. 2001).

4.2 Optimization ofCulture Conditions

Optimizing culture conditions is an effort to optimize the conditions (external and

internal) of invitro culture so that maximum production (biomass and secondary

metabolites) is obtained (Bienaimé et al. 2015). Optimization of tissue culture

media is useful in applied research and engineering of secondary metabolite biosyn-

thetic pathways (Heidargholinezhad etal. 2023) to produce biomass and secondary

metabolite production on a large scale (Bienaimé etal. 2015). The culture media are

easy to manipulate by changing the sugar composition, type, and concentration of

plant growth hormones, phosphate, and nitrogen levels (Zhong 2001). Increasing

knowledge about economically important plant secondary metabolite pathways has

implications for increasing the use of plant cultures to produce bioactive compounds

(Vanisree etal. 2004).

Optimum culture medium and environmental factors are the basic approaches

that must rst be addressed to increase secondary metabolite production (Murthy

etal. 2014; Isah etal. 2018; Raj and Saudagar 2019). Culture conditions that need

to be optimized to increase secondary metabolites include nutrient media composi-

tion, inoculum density, temperature, light, agitation, and aeration (Wawrosch and

Zotchev 2021; Murthy etal. 2014). Optimal levels of carbohydrates, organic com-

pounds (vitamins), mineral nutrients, environmental factors (e.g., light, gaseous

environment, temperature, and humidity), and growth regulators are required to

obtain high regeneration rates in commercial MPs (Rout etal. 2000).

M. Silalahi et al.

263

Manipulation of media components is one way to increase secondary metabo-

lites, so that expressing the necessary secondary metabolite pathways (Raj and

Saudagar 2019; Zhou et al. 2009) can be conducted by selecting media, carbon

sources, and growth regulators (Mahmood et al. 2021; Delcheh et al. 2014).

Metabolic engineering offers a new perspective to increase the production of desired

compounds (Verpoorte etal. 2002). The shoot regeneration on Lippia citriodora

inoculated onto Murashige and Skoog’s media supplemented with indole-3-acetic

acid (IAA) is better than 6-benzylaminopurine (BAP) and Kinetin (Kn) (Delcheh

etal. 2014). Indirect organogenesis of Lallemantia royleana with the best results

(100%) was obtained with a combination of BAP (1.0mg/L) and IAA (2.0mg/L).

Shoot formation was achieved by administering BAP (1.0mg/L) and Kn (0.5mg/L),

while the best rooting was achieved with naphthalene acetic acid (NAA 1mg/L)

(Mahmood etal. 2021).

Growth regulators (GH) are the most important factors inuencing cell growth,

differentiation, and secondary metabolite production (Bienaimé et al. 2015). In

Centella asiatica culture, the types of GH in the form of BAP (2.0mg/L) and NAA

(0.1mg/L) on MS media induce optimal shoots (Siavash etal. 2011), while opti-

mum rooting was obtained at 0.5 mg/L (Siavash et al. 2011)–1mg/L indole-3-

butyric acid (IBA) (Heidargholinezhad etal. 2023). The GH interactions greatly

inuence the biomass of Lycopodiella inundata cell culture which increases by ve

times, and the alkaloid content reaches 1% (alkaloid weight/DW) under optimal

conditions (Bienaimé etal. 2015).

4.3 Elicitation

The concentration of secondary metabolites produced naturally by plants is quite

low and will increase if they experience stress. Elicitation is an increase in biomass

production and secondary metabolites in plants invitro and ex vivo (Nabi etal.

2021; Halder etal. 2019), undifferentiated or differentiated cultures (Halder etal.

2019). Plant defense responses can be induced by elicitors (Sharma and Shahzad

2013) thus stimulating increased production of secondary metabolites especially in

invitro cultures (Kandoudi and Németh-Zámboriné 2022). Elicitors act as signals,

recognized by elicitation-specic receptors on plant cell membranes and stimulate

defense responses during elicitation (Halder etal. 2019) as well as affect different

biochemical and molecular pathways (Nabi etal. 2021) during elicitation resulting

in increased synthesis and accumulation of secondary metabolites (Halder etal.

2019; Nabi etal. 2021).

In general, elicitors can be divided into abiotic and biotic (Zhao etal. 2010;

Sharma and Shahzad 2013; Halder etal. 2019) and physical and chemical (Sharma

and Shahzad 2013) elicitors, but the abiotic and biotic grouping is more widely used

by experts. Abiotic elicitors can be heavy metal ions (Co2+, Ag+, Cd2+), chemical

compounds (salicylic acid, methyl jasmonate), and hyperosmotic stress (sorbitol)

(Zhao etal. 2010). Biotic elicitors can be yeast extracts (Silalahi 2010; Zhao etal.

Biotechnology Toward Medicinal Plants (MPs)

264

2010) and fungal extracts either in the form of crude extracts or in polysaccharides

(Zhao etal. 2010; Li etal. 2011; Zhang etal. 2009). Elicitors consist of different

compounds between oligosaccharides or lipo and protein glycols. These biotic elici-

tors are often derived from pathogens (exogenous elicitors) but in some cases are

released from plants attacked by the action of pathogenic enzymes (endogenous

elicitors) (Sharma and Shahzad 2013). Combinations of different elicitor applica-

tions, integration of precursor feeding, or addition of media or in situ, product

recovery from roots/liquid media with elicitor treatments have shown increased

accumulation of secondary metabolites due to their synergistic effects (Halder

etal. 2019).

The increase in secondary metabolite concentration due to the addition of elici-

tors varies greatly inuenced by various factors both internal and external (such as

elicitation concentration, duration, age, and culture composition (Nabi etal. 2021).

Halder etal. (2019) stated that optimization of various parameters, such as the type

of elicitation, concentration, duration of exposure, and time of administration, is

crucial for the effectiveness of the elicitation strategy. Elicitation of Gingko biloba

cell culture with methyl jasmonate (MJ) and salicylic acid (SA) in immobilized

cells increased bilobalide and ginkgolides A, B, and C, compared to the unelicited

control (Sukito and Tachibana 2016).

In fact, elicitors sometimes increase certain types of metabolites but on the other

hand increase other types (Hashem 2018; Boroduske etal. 2016), and the degree of

increase also varies between bioactive compounds (Li etal. 2011; Inyai etal. 2019).

Hair root cultures are preferred for elicitation application due to their genetic and

biosynthetic stability, high growth rate in growth regulator-free medium, and con-

sistency of production in response to elicitation treatment (Halder et al. 2019).

Foliar application of salicylic acid (SA) elicitors (0.724 and 1.448mM) signi-

cantly increased the percentage of essential oil by 100% in Majorana hortensis.

Elicitation with 0.724 mM concentration increased a-terpinene, c-terpinene,

a- terpinolene, and terpinen-4-ol percentages but decreased the sabinene and

4- thujanol percentages by 26% and 57%, respectively (Hashem 2018). The effect of

elicitation yeast extract, methyl jasmonate (MeJA), and chitosan on the yield of

secondary metabolites showed that elicitation with MeJA and chitosan signicantly

decreased the yield of sweroside and eustomin in Centaurium erythraea invitro

shoot, callus, and cell suspension cultures (Boroduske etal. 2016).

Although the administration of elicitors can increase the production of secondary

metabolites, it sometimes results in decreased cell growth (Zhao etal. 2010), due to

the accumulation of phenolic compounds (Dias etal. 2016). Elicitation procedures

are often used to increase phenolic production, in many cases achieving higher

yields than non-elicited cultures (Dias et al. 2016). Most elicitors suppress the

growth of Salvia miltiorrhiza cell cultures, decreasing biomass yield by about 50%.

Elicitors stimulated cell phenylalanine ammonia lyase activity and transient

increases in medium pH and conductivity (Zhao etal. 2010). In Vitis vinifera cul-

tures, chitosan treatment signicantly increased hydrogen peroxide and phenylala-

nine ammonia lyase (PAL) production (Dörnenburg 2004). Table 2 shows the

increase of various secondary metabolites with the addition of bioactive and abiotic

elicitors.

M. Silalahi et al.

265

4.4 Feed Precursor

The exogenous or endogenous compounds that are converted by cultured plant cells

into secondary metabolites through the biosynthetic pathway are called precursors

(Isah etal. 2018). The primary metabolic process produces various intermediate

compounds (Fig.1) which become precursors (“basic materials”) for the formation

of secondary metabolites; therefore, the availability of precursors directly or indi-

rectly inuences the content of the nal product of secondary metabolites (Patil

etal. 2013; Vu etal. 2022). The addition of precursors to the biosynthesis pathway

is considered an effective approach for the formation of secondary metabolites

(Watcharatanon etal. 2019; Isah etal. 2018) and allows the production of new sec-

ondary metabolites that are not produced without the introduction of precursors

(Verpoorte etal. 2002). The addition of precursors develops based on the premise

that compounds between bioactive compound molecules when added at the begin-

ning or during the culture period function as additional substrates to increase the

production of high metabolites in plant cells (Isah etal. 2018).

An increase in the secondary metabolite content of MPs with the addition of

precursors has been reported in invitro cultures of Bacopa monnieri (Watcharatanon

etal. 2019), Catharanthus roseus (Vu etal. 2022), Digitalis purpurea (Patil etal.

2013), and Morus alba (Inyai etal. 2019). Administration of l-alanine (5mM) and

l-phenylalanine (150μM) precursors to Bacopa monnieri (MPs to reduce anxiety)

culture signicantly increased the accumulation of triterpenoid saponin glyco-

sides increasing up to 2.6-fold after 6days (Watcharatanon etal. 2019). The addi-

tion of certain precursors (L-phenylalanine, L-tyrosine) to the hairy roots of

C. roseus (an anticancer plant) was able to produce vincristine (51.99μgg−1 DW),

and vinblastine (699.92μgg−1 DW) was obtained in seventh week (with biomass

0.306 g DW) (Vu et al. 2022). Administration of progesterone precursor

(200–300mg/L) to D. purpurea shoot cultures increased the accumulation of digi-

toxin and digoxin by 9.1- and 11.9-fold, respectively (Patil et al. 2013).

L-phenylalanine (0.05 mM), L-tyrosine (0.03 mM), or a combination of both

resulted in an increase in mulberroside A production up to twofold in M. alba cell

suspension cultures (Inyai et al. 2019). The added L-tyrosine signicantly

increased the production of oxyresveratrol and resveratrol of M. alba cell suspen-

sion culture (Inyai etal. 2019).

4.5 Cell Immobilization

Cell immobilization is the process of xating (trapping) plant cells on trapping

materials (agar, agarose, calcium alginate, or carrageenan) with the aim of increas-

ing the production of plant secondary metabolites (Nartop 2016; Brodelius and

Mosbach 1982). Techniques for plant cell immobilization originated from those

used to trap enzymes and microbial cells and in principle have utilized a wide vari-

ety of gel materials (Lindsey etal. 1983). Immobilized cells are deposited on the

Biotechnology Toward Medicinal Plants (MPs)

266

Table 2 Several applications of abiotic and biotic elicitors in the production of plant secondary metabolites

No. Scientic name Elicitor Tissue culture type Secondary metabolites Result References

1Allium sativum L. Fe3+ and Zn2+ Callus culture of crown

explants in MS medium

with 2,4-D growth

regulators and kinetin

Organosulfur compound,

including alliin, allicin,

ajoene

Fe3+ and Zn2+ elicitors signicantly

increased organosulfur compounds in

single garlic cell culture, with the

highest enhancements seen at

0.3mM Zn2+ (twofold) and 0.5mM

Fe3+ (1.5-fold)

Setiowati etal.

(2022)

2Angelica

archangelica

(C.B.Clarke)

Weinert

Aspergillus niger

extract

Callus induction of leaf

explants in MS medium

with 2,4-D growth

regulators and kinetin.

Cell suspension culture

from callus gown in

liquid nutritional

medium

Coumarin The use of A. niger extract as elicitor

in A. archangelica culture,

specically at 2.0mL/L for 21days

increased coumarin content by

twofold compared to control group

Nadir (2022)

3Aquilaria laria Yeast extract and

laminarin, silver

nitrate and copper

sulfate

In vitro cultures of

plantlet leaf explants in

MS medium with

sucrose, MES, BAP,

and IBA, agar

Volatile fragrance

sesquiterpenes such as

jinkohol and acorenone

In vitro cultures of A. laria

exhibited a high inducibility to

produce various aromatic

sesquiterpenes compounds, which

are recognized as key components

contributing to the fragrance of

agarwood

Listiana etal.

(2021)

4Aquilaria

malaccensis Lam.

Crude extracts of

Fusarium solani

strains, methyl

jasmonate (MeJA)

Callus culture of leaf

explants in MS medium

with 2,4-D and BA

growth regulators

Alkenes including

1-docosene and

1-octadecene, phenolic

(4-ditetra-buthylphenol),

and fatty acid

(benzenepropanoic acid)

The treated calli exhibited increased

production of fatty acid derivatives

compared to the control group. The

elicitors employed in this study

effectively stimulated the synthesis

of agarwood-related compounds,

including chromone and fatty acids,

in the callus culture

Faizal etal.

(2023)

M. Silalahi et al.

267

5Aquilaria

malaccensis Lam.

Crude extracts of

Fusarium solani

strains, MeJA

Callus culture of

axillary bud explants in

MS medium with BA

growth regulators

Sesquiterpenes and

chromone derivatives

(agarwood compounds)

Agarwood compounds, including

sesquiterpenes and chromone

derivatives, were detected in

MeJA-treated shoots. Moreover,

extracts from F. solani induced the

presence of alkanes, aromatic

compounds, and fatty acid derivatives

Faizal etal.

(2021)

6Camellia sinensis

Cobalt (II) chloride

ionic

Callus culture of leaf

shoots explants in MS

medium with 2,4-D

growth regulators

Cinnamic acid The addition of cobalt (II) chloride

ionic as elicitor resulting in an

increase of cinnamic acid by 11.9%

Sutini etal.

(2019)

7Camellia sinensis

Phosphor In vitro culture of leaf

shoots explants in MS

medium with 2,4-D

growth regulators and

optimized by phosphor

inducer

Epicatechin The addition of phosphor inducers

during invitro culture of C. sinensis

resulted in a signicant 11.75%

increase in epicatechin, representing

a substantial improvement compared

to previous results

Sutini etal.

(2020)

8Carica papaya L. Saccharomyces

cerevisiae

In vitro culture in MS

medium and VW

medium with NAA,

BAA, and glucose

Papain S. cerevisiae boosted callus

formation and increased papain

content, especially when combined

with VW medium and 25mg dry

matter/L of S. cerevisiae as an

elicitor, resulting in a 0.020% rise in

papain content compared to the MS

medium control

Suryaningsih

etal. (2020)

(continued)

Biotechnology Toward Medicinal Plants (MPs)

268

Table 2 (continued)

9Chrysanthemum

morifolium Ramat

Sucrose Callus culture in MS

medium with 2,4-D

growth regulators and

different sucrose

concentration

Quercitrin, including

quercetin-3-O-rhamnoside

The treatment with 45g/L of sucrose

and a 30-day harvest yielded the

highest fresh weight, dry weight, and

quercitrin content: 2.108g, 0.051g,

and 0.437mg/g DW, respectively.

Notably, quercitrin content

demonstrated a 63.67% increase

compared to the control

Setiawati etal.

(2023)

10 Chrysanthemum

morifolium Ramat

Polyethylene

glycol (PEG)

In vitro culture of leaf

explants in MS medium

with BAP

Quercitrin The addition of 10ppm PEG to the

shoots culture resulted in the highest

quercitrin content, showing a

343.55% increase compared to the

control. As the PEG concentration

increased, quercitrin levels declined

Setiawati etal.

(2019)

11 Cinchona

ledgeriana

(Howard) Bern.

Moens ex Trimen

ABA, sucrose Cell suspension culture

of leaves and stem bark

explants with A3K

treatment

Quinine The use of ABA (3mg/L) and a

partial replacement of sucrose with

sorbitol as elicitors over a 7-week

period (A3S7) led to a doubling in

quinine production compared to the

4-week treatment (A3S4)

Ratnadewi

etal. (2021)

12 Citrus hystrix DC. Yeast extract,

isopentenyl

pyrophosphate

In vitro culture of seed

explants in MS medium

with 2,4-D growth

regulators

Bioactive compounds,

including alpha-

humulene, nerolidol,

farnesol, beta-carotene,

phytol, squalene

The increase in bioactive compounds,

specically terpenoids in C. hystrix

ranged from 0.39 to 5.11% compared

to the control group when using yeast

and isopentenyl pyrophosphate as

elicitors

Damayanti

etal. (2023)

No. Scientic name Elicitor Tissue culture type Secondary metabolites Result References

M. Silalahi et al.

269

13 Citrus hystrix DC. Alginate

encapsulation

(alginate solution

and CaCl2.2H2O

solution 0.2M)

In vitro culture of seed

explants in MS medium

with 2,4-D and sodium

salt monohydrate

Squalene, geranyl

linalool, and geranyl

acetate

Storage at 4°C, with or without

encapsulation, altered the prole of

bioactive compounds. Terpenoids

like squalene, geranyl linalool, and

geranyl acetate were detected after

preservation. Some anticancer

bioactive compounds were also

detected including stearic acid.

Fajarina etal.

(2021)

14 Curcuma mangga

Val.

Yeast extract and

chitosan

Callus culture of shoot

explants on MS

medium with BA and

NAA

Anti-lipid peroxidation

activity

C. mangga, treated with various

elicitors, demonstrated signicant

anti-lipid peroxidation activity,

exhibiting a notable increase ranging

from approximately 67% to 70%

compared to the negative control of

absolute ethanol

Abraham etal.

(2022)

15 Glycine max (L.)

Merr.

Saccharomyces

cerevisiae, yeast

extract, light

Callus culture from

seed explants and

suspension culture

Glyceollin Ragi tape with light (gRL) increased

glyceollin I content by sevenfold

compared to untreated soybeans,

showing superior efcacy over S.

cerevisiae with light (gSL)

Athoillah etal.

(2019)

16 Glycine max (L.)

Merr.

Pulsed electric

eld (PEF)

Not mentioned Phenol, avonoid, tannin,

antioxidant activity

The use of PEF with thermocontrol

as an elicitor in soybeans, specically

at conditions of 3kV and 25°C,

resulted in increased total of phenols

by 5.8%, avonoids by 19.4%,

tannins by 5.3%, and antioxidant

activity by 79.5% compared to the

control

Maligan etal.

(2020)

(continued)

Biotechnology Toward Medicinal Plants (MPs)

270

Table 2 (continued)

17 Medinilla

verrucosa Bl.

GA3 and PGPRs

(plant growth

promoting

rhizobacteria)

Soil-based system Antioxidant content

(phytoalexins)

The increase of antioxidant activity

in M. verrucosa ranged from 11.7 to

51.4% compared to the control group

when GA3 and PGPRs as elicitors

Sakya etal.

(2022)

18 Morinda citrifolia

(L.)

Chitosan shell of

shrimps (Penaeus

monodon)

Callus culture of leaf

explants in MS medium

with 2,4-D growth

regulators and kinetin

Anthraquinone The addition of 2mg/mL chitosan as

an elicitor, with a 4-day elicitation

time, resulted in a remarkable

increase in anthraquinone content in

M. citrifolia callus culture, showing a

signicant rise of 323.75% or

approximately threefold compared to

the control

Purwianingsih

etal. (2019)

19 Panax ginseng Yeast extract,

coconut water

Hairy root culture Ginsensoside The initial addition of 20mg/L yeast

extract did not elevate ginsenoside

levels in P. ginseng hairy root culture.

Similarly, using 10mL of coconut

water from the beginning increased

biomass but did not affect

ginsenoside levels in the culture

Sukweenadhi

etal. (2023)

20 Physalis angulata

Chitosan,

polyethylene

glycol (PEG)

In vitro culture of shoot

explants in MS medium

Withanolides Chitosan elicitor shows more

promise than PEG as it enhances

withanolides content with less

growth inhibition. In contrast, PEG

elicitor hinders shoot growth more

signicantly with a comparatively

lower increase in withanolides

content

Mastuti and

Rosyidah

(2019)

No. Scientic name Elicitor Tissue culture type Secondary metabolites Result References

M. Silalahi et al.

271

21 Physalis angulata

Chitosan In vitro culture of shoot

induction on MS

medium with BAP and

IAA

Withanolides Chitosan elicitor increased

withanolide levels in P. angulata

invitro shoots. Among the

accessions, A4 demonstrated the

highest increase in withanolide

content, reaching 108.97%

Mastuti etal.

(2021)

22 Piper betle L. Cobalt (II) chloride Callus culture of leaf

explants in MS medium

with 2,4-D growth

regulators and BAP

Terpenoids The addition of 1.0mg/L CoCl2 at

2weeks of age led to an increase in

terpenoids content to 5.95%,

compared to the control. Among the

terpenoids identied, the two most

prominent types were 1,2-epoxy-1-

vinylcyclododecene and

hexadecanoic acid

Junairiah etal.

(2020)

23 Talinum

paniculatum

(Jacq.) Gaertn.

MeJA Callus culture of leaf

explants in MS medium

with sucrose, agar,

2,4-D, and kinetin

Saponin The addition of 0.15mM MeJA to

the callus culture for 15days led to a

70.6% increase in saponin content,

demonstrating its potential for

enhanced saponin production

Restiani etal.

(2022)

24 Talinum

paniculatum

Gaertn.

MeJA and salicylic

acid

In vitro culture of

adventitious roots on

MS medium with

indole-3-butyric acid or

IBA

Saponin Elicitation with 0.2mM MeJA and

SA for 15days resulted in a

respective 1.5- and 1.3-fold increase

in saponin production. This

highlights that MeJA and SA

treatments modulate saponin

biosynthesis in T. paniculatum

adventitious root culture, with effects

dependent on both time and dosage

Faizal and Sari

(2019)

(continued)

Biotechnology Toward Medicinal Plants (MPs)

272

Table 2 (continued)

25 Trigonella

foenum-graecum

Sodium chloride

(NaCl) and lysine

Callus induction of

shoot explants on MS

medium with NAA and

Proline and trigonelline The addition of NaCl (from 0 to

8g/L) led to an approximate 15.70%

increase in proline content and a

signicant 97.40% increase in

trigonelline content. On the other

hand, the introduction of lysine (from

0 to 40mg/L) resulted in a 56.20%

rise in proline content and a

noteworthy 90.00% increase in

trigonelline content

Madhloom

etal. (2020)

26 Vetiveria

zizanioides

L.Nash

Lead (Pb) and

cadmium (Cd)

In vitro culture of

adventitious roots on

MS medium with

kinetin, NAA

Vetiver oil compounds,

including khusimone,

khusimol, khusimene,

α-vetivone,β-vetivone,

vetiverol, prezizaene,

zizaene

The elicitation treatments with lead

(Pb) and cadmium (Cd) resulted in

notable increases in specic

compounds within vetiver oil. Across

various compounds, Pb elicitation led

to an average increase of

approximately 3.5%, while Cd

elicitation showed a slightly higher

increase of around 5.5%

Ivani and

Widoretno

(2019)

M. Silalahi et al.

273

surface of articial brous materials that provide a strong binding making them

immobile to plant tissue biomass grown in submerged culture (Archambault etal.

1989). Immobilized plant cells maintain prolonged survival and biosynthetic capac-

ity with high sustained secondary levels of metabolite production (Archambault

et al. 1989; Singh and Kaur 2014). Immobilization can be an effective tool to

increase the effectiveness of plant cell production processes (Dörnenburg 2004).

The most frequently used immobilization methods include gel encapsulation, sur-

face immobilization, and entrapment by membrane barriers (Singh and Kaur 2014).

When compared to other techniques, plant cell immobilization is a new technol-

ogy but has excellent potential to increase phytochemical production (Rosevear and

Lambe 2005) and is an attractive alternative to chemical synthesis or natural har-

vesting of high-value secondary metabolites in the pharmaceutical industry (Singh

and Kaur 2014). Various researchers have successfully demonstrated the enhance-

ment of secondary metabolites used as drugs through immobilization such as

Tinospora cardifolia (Roja etal. 2005), Ginkgo biloba (Sukito and Tachibana 2016),

Vitis vinifera, Cruciata glabra (Dörnenburg 2004), Morus alba (Inyai etal. 2019),

Solanum chrysotrichum (Charlet et al. 2000), and Lithospermum erythrorhizon

(Kim and Chang 1990). As immobilized cells are in direct contact with the nutrient

medium, there is no permeability barrier to nutrients and metabolites that can be

created by the gel (Lindsey etal. 1983).

The increase in secondary metabolite content through immobilization varies

greatly inuenced by various factors including culture conditions (Roja etal. 2005)

and type of trapping material (Komaraiah etal. 2023; Roja etal. 2005). Immobilized

Tinospora cordifolia callus cultures (sodium alginate and calcium chloride and the

beads), when cells were subjected to elicitation and permeabilization with chitosan

and in situ removal of secondary metabolites by resin addition, showed a tenfold

increase in arabinogalactan production (0.490% dry weight) compared to controls

without resin and chitosan (Roja etal. 2005). Bilobalide and ginkgolides content in

biomass-immobilized Ginkgo biloba cell suspension cultures was approximately

1.4 times higher than in cell suspension cultures, and the production of bilobalides

and ginkgolides A, B, and C increased 5.0, 3.3, 6.1, and 4.1 times, respectively

(Sukito and Tachibana 2016). In Cruciata glabra, the immobilization process

increased hydrogen peroxide synthesis by 533% followed by an increase in anthra-

quinone concentration by 556% (Dörnenburg 2004). Plumbago rosea cell cultures

immobilized in calcium alginate increased plumbagin production by three-, two-,

and onefold compared to control, non-crosslinked alginate-treated cells and CaCl2

cells, respectively. The Plumbago rosea cells immobilized at a level of 20% to the

volume of polymer (Na-alginate) was optimal, and maximum plumbagin was

obtained, while higher loading of cells (40–50%) and lower accumulation of plum-

bagin were seen (Komaraiah etal. 2023).

Immobilized cell culture has advantages compared to other methods such as

some of the metabolites are excreted into the media (Charlet etal. 2000) thus facili-

tating their harvesting (Inyai etal. 2019; Charlet etal. 2000; Komaraiah etal. 2023).

Ca-alginate gel beads immobilized Solanum chrysotrichum cell suspense culture

secreted the saponin spirostanol into the surrounding medium (about 40% of total

Biotechnology Toward Medicinal Plants (MPs)

274

production) (Charlet etal. 2000). More than 70% of plumbagin is released into the

medium, which is highly desirable for easy recovery of Plumbago rosea cell culture

products (Komaraiah etal. 2023). Alginate immobilization of M. alba cell suspense

culture signicantly increased the secretion of mulberroside A into the extracellular

matrix and culture medium by up to 60%, while increasing the levels of oxyresve-

ratrol and resveratrol by 12- and 27-fold, respectively (Inyai et al. 2019).

Immobilization of Lithospermum erythrorhizon cell culture with calcium alginate

gave higher specic shikonin productivity by 2.5 times, respectively, compared with

free cell culture (Kim and Chang 1990).

The increase in secondary metabolite content was higher in cell cultures immo-

bilized together with elicitors (Komaraiah etal. 2023). The addition of 200mgL−1

chitosan as an elicitor to immobilized Plumbago rosea cells resulted in eight- and

twofold higher accumulation of plumbagin compared to control and immobi-

lized cells (Komaraiah etal. 2023). Cells treated with a combination of chitosan,

immobilization, and in situ extraction showed a synergistic effect and produced

92.13 mg g−1 DCW plumbagin which was 21, 5.7, and 2.5 times higher than

control, immobilized, immobilized, and elicited cells, respectively (Komaraiah

etal. 2023).

5 Bioreactors forLarge-Scale Production

ofSecondary Metabolites

The large-scale production of secondary metabolites/bioactive compounds from

MPs holds signicant benets for the pharmaceutical industry (Rahmat and Kang

2019). Bioreactors offer a promising option for the large-scale production of sec-

ondary metabolites from MPs, with several signicant advantages (Luthra etal.

2022; Rohini and Rajasekharan 2022). They enable optimal environmental control,

including temperature, pH, aeration, and nutrition, ensuring ideal conditions for

secondary metabolite production (Murthy etal. 2012; Natanael etal. 2014; Komaikul

etal. 2019; Ozyigit etal. 2023). Various bioreactor congurations, including liquid-

phase reactors, gas-phase reactors, and hybrid reactors, have been proposed for

plant culture, each with distinct advantages and growth conditions contributing to

overall secondary metabolite production efciency (Thakore and Srivastava 2020;

Mitra and Murthy 2022). Bioreactors can also be adapted to various scales, allowing

large-scale production for the pharmaceutical industry (Kowalczyk etal. 2022). The

cultivation of MPs cells in bioreactors follows the basic principle of isolating plant

cells and growing them in a controlled environment with appropriate nutrients and

growth factors (Wawrosch and Zotchev 2021).

For commercial-scale production, the various stages of secondary metabolites/

bioactive compounds production from MPs can be seen in Fig.3. Table3 highlights

the utilization of bioreactors for commercial production. The enhancement of sec-

ondary metabolites/bioactive compounds, as discussed in the previous subsection,

M. Silalahi et al.

275

Fig. 3 Production of secondary metabolites from medicinal plants and their stages using bioreactors (modication from Wawrosch and Zotchev 2021)

Biotechnology Toward Medicinal Plants (MPs)

276

Table 3 The table below presents publications related to the utilization of bioreactors in the production of secondary metabolites from medicinal plants in

Indonesia over the last 5years

No.

Scientic

name

Plant part

used

Secondary

metabolites Bioreactor Uses References

1Aquilaria

malaccensis

Shoot Renin 0.5L bubble column

bioreactor and temporary

immersion system (TIS)

RITA bioreactor

Abdominal complaints, asthma, colic, diarrhea,

aphrodisiac, carminative

Esyanti etal.

(2019)

2Citrus

limonia

Callus Limonin Airlift bioreactor Antitumor, neuroprotective, immunomodulatory,

analgesic, anticancer agent against liver, breast, and

colon cancers, as well as for anti-inammatory,

antibacterial, antioxidant, antiviral, antimalarial,

and larvicidal activities, obesity, hyperglycemia,

nonalcoholic fatty liver, cardiovascular diseases,

type 2 diabetes, and metabolic syndromes

Agisimanto

etal. (2022)

3Gynura

procumbens

(Lour.)

Merr.

Root Kaempferol,

quercetin

Balloon-type bubble

bioreactor (BTBB) with

phenylalanine

Fever, rash, hypertension, migraine, constipation,

kidney problems, diabetes mellitus, and cancer

Manuhara

etal. (2019)

Adventitious

root

Myricetin,

catechin,

quercetin,

kaempferol

3L capacity BTBB with

optimization of aeration

volume and inoculum

density

Cancer, arteriosclerosis, and cardiovascular

treatment, antiviral and antidiabetic properties

Kusuma etal.

(2021)

Adventitious

root

Kaempferol,

quercetin

19L capacity airlift

BTBB

Cancer, arteriosclerosis, and cardiovascular

treatment, antiviral and antidiabetic properties

Kusuma etal.

(2023a)

Root Kaempferol,

quercetin

3L capacity airlift BTBB Cancer, arteriosclerosis, and cardiovascular

treatment, antiviral and antidiabetic properties

Kusuma etal.

(2023b)

Shoot Quercetin,

kaempferol

BTBB in different

sucrose concentration and

varieties of inoculum

density

Antihyperglycemic, antihypertensive, antimicrobial,

antioxidant, anti-inammatory, anticancer,

cardioprotective, and increasing fertility

Saadah etal.

(2022)

M. Silalahi et al.

277

No.

Scientic

name

Plant part

used

Secondary

metabolites Bioreactor Uses References

4Musa

acuminata

Shoot Gallic acid Bubble column

bioreactor

Antioxidant, anti- inammatory, and antineoplastic

properties

Nurhayati

etal. (2022)

5Panax

ginseng

C.A.Mey

Root Ginsenosides 18L BR-BIO180

bioreactor with various

inoculum weights

Increasing blood circulation, antioxidants, blood

pressure, and tumor suppression

Chandra etal.

(2021)

Adventitious

root

Ginsenosides 18L BR-BIO180

bioreactor with increase

in media volume

Antioxidant, anti- inammatory, antiallergic,

antidiabetic, anticancer, to treat neurodegenerative

diseases

Natalie etal.

(2022)

6Stevia

rebaudiana

Berth.

Shoot Steviol

glycosides

(stevioside)

Temporary immersion

bioreactor (TIB) with

daminozide treatments

Antidiabetic Saptari etal.

(2022)

Shoot Stevioside,

rebaudioside-A

Temporary immersion

system (TIS) RITA

bioreactor induced with

high far-red LED light

Antidiabetic Melviana

etal. (2021)

Biotechnology Toward Medicinal Plants (MPs)

278

can also be applied in bioreactors (Saraswati 2012; Ramirez-Estrada etal. 2016).

This approach allows for larger and more controlled production compared to con-

ventional culture (Carlo etal. 2021; Agisimanto etal. 2022).

The use of bioreactors also presents several challenges and risks, including con-

tamination, difculties in optimizing environmental conditions, and complex mass

transfer (Georgiev etal. 2013). To address these issues, precise sterilization, the use

of sensors and automated control systems, careful bioreactor design, and environ-

mental monitoring around the bioreactor are essential (Connelly et al. 2017;

Saraswati 2012).

6 Conservation ofMedicinal Plants (MPs) Through

InVitro Technology

Currently, the rate of species extinction has caused around 34,000 of the 270,000

existing plant species to be included in the endangered category. In terms of MPs,

which play an important role in traditional and modern medical practices, around

80% of them are still collected from wild populations that grow naturally (Sharma

etal. 2020). For example, many MPs species in Indonesia are harvested destruc-

tively (Cahyaningsih etal. 2021) by removing rootlets (roots, rhizomes, or tubers)

or bark or by harvesting the entire plant (Schippmann etal. 2002). These destruc-

tive harvesting methods increase the risk of growth failure or even plant death

(Volenzo and Odiyo 2020a, b). Overexploitation, overharvesting, and degradation

of natural habitats have threatened the survival of many MPs species (Roberson

2008; Hawkins 2008; Luthra etal. 2022). This has resulted in the extinction of

many MPs (Alamgir and Alamgir 2017) and the loss of genetic diversity (Rather

etal. 2022). Some of the endangered MPs are Saussurea lappa, Picrorhiza kurroa,

Ginkgo biloba, Swertia chirata, Gymnema sylvestre, Tinospora cordifolia, Salacia

oblonga, Holostemma, Celastrus paniculatus, Oroxylum indicum, Glycyrrhiza gla-

bra, Tylophora indica, Bacopa monnieri, and Rauwola serpentina (Sharma etal.

2010). To prevent plant extinction, it is necessary to develop conservation strate-

gies (Sarwat et al. 2012), especially for step and endangered MPs (Sharma

etal. 2010).

In vitro technology offers a variety of approaches to address the challenges of

MPs conservation (Kowalczyk etal. 2022). In vitro technology has advantages over

conventional conservation, including (1) enabling long-term storage of MPs genet-

ics through seed cryopreservation (Anuruddi etal. 2023), (2) maintaining genetic

diversity, (3) preventing the extinction of superior varieties (Chandran etal. 2020),

(4) developing superior varieties (Grzegorczyk-Karolak etal. 2021), and (5) creat-

ing new genetic variations in breeding strains (Brown and Thorpe 1995). In vitro

techniques for the culture of protoplasts, anthers, microspores, ovules, and embryos

have been used to create new genetic variation in breeding lines, often through the

production of haploids (Brown and Thorpe 1995). Kulak etal. (2022) asserted that

M. Silalahi et al.

279

there are three main applications of invitro technology in conservation: (1) invitro

methods facilitate the utilization of the ability of plant tissues to reproduce vegeta-

tively from the limited starting material, thereby reducing the need to harvest whole

plants or many plants from nature, and preventing the depletion of vulnerable popu-

lations in their natural habitats; (2) invitro-grown specimens of threatened or rare

plants can also help replenish ex situ collections in botanical gardens and other

research institutions that can later be reintroduced in natural habitats; and (3) these

techniques allow the production of unlimited quantities of explants to supply mate-

rial for scientic experiments.

In vitro culture can be used as an alternative strategy for the conservation of

endangered genotypes, which can be achieved by using different growth media. It

can help to maintain the genetic background of plants and avoid loss of conserved

heritage due to environmental changes, and stresses, both biotic and abiotic (Rather

et al. 2022). In vitro culture has also been reported to produce somaclonal and

gametoclonal variants with crop improvement potential (Brown and Thorpe 1995).

For the sterile plants, it can be preserved through invitro cold storage and cryo-

preservation in liquid nitrogen at very low temperatures of −96°C (−320 °F)

(Marthe 2018; Kundu et al. 2018; Rajasekharan and Prakashkumar 2010). This

technique can be used on a large scale for a wide variety of materials, including

seeds with orthodox and intermediate storage behavior, dormant buds, pollen, bio-

technology products, and apical tips taken from vegetatively propagated invitro

plantlet species (Rajasekharan and Prakashkumar 2010).

The methods of cryopreservation techniques, such as encapsulation-dehydra-

tion, vitrication, encapsulation-vitrication, and droplet vitrication, can pre-

serve many MPs species for future generations (Tahtamoun et al. 2015;

Rajasekharan and Prakashkumar 2010). These methods can guarantee the supply

of plant tissue, genetic stability, and retention of biosynthetic potential, which is

sustainable for a long time (Tahtamoun etal. 2015; Mosa etal. 2023; Rajasekharan

and Prakashkumar 2010). Tahtamoun etal. (2015) stated that cryopreservation, a

method of long-term storage for germplasm, is more protable than most other

conservation methods because of its simplicity, easy of application in various

genotypes, and the ability to maintain the genetics and stability of plant material.

Different cryopreservation methods are used for long-term cultural preservation,

which allows rapid regeneration of preserved plant material and maintain the origi-

nal properties (Mosa etal. 2023). One of the important things that need to be con-

sidered in plant conservation through cryopreservation is the level of recovery and

genetic stability of plant tissue (Mosa etal. 2023). The morphology of plants regen-

erated from cryopreserved callus is similar to the morphology of seed plants (Sen-

Rong and Ming-Hua 2012). After cryopreservation, the young shoots of Picrorhiza

kurroa have an average survival of approximately 20% without callus formation

(Sharma and Sharma 2003). Cold hardening of shoot cultures for 4weeks at 4°C

increased survival and shoot regeneration from cryopreserved shoot tips by 70%

and 35%, respectively (Sharma and Sharma 2003). Table4 shows the use of cryo-

preservation for MPs that have commercial value.

Biotechnology Toward Medicinal Plants (MPs)

280

Table 4 Conservation of medicinal plants using cryopreservation techniques

No. Scientic name Secondary metabolites Uses

Types of culture, medium,

and supplements Techniques References

1Anemarrhena

asphodeloides

Bunge

Xanthone C-glycoside, mangiferin Febrile diseases, fever,

cough, and diabetes

Callus embryonic, MS

medium with 2mg/L Kn,

0.1mg/L α- NAA, and

0.5M sucrose

Vitrication Sen-Rong and

Ming-Hua (2012);

Huang etal.

(2022)

2Byrsonima

intermedia

A.Juss

Digalloyl quinic acid, galloyl

quercetin hexoside,

galloylproanthocyanidin dimer,

proanthocyanidin dimer, quercetin-

O- hexoside, galloyl quercetin

pentoside, and

quercetin-O-pentoside

Gastrointestinal,

inammations, skin

infections,

gynecological and

snakebites

Shoot tip, woody plant

medium with 2.22μM BAP

Droplet

vitrication

Coutinho Silva

etal. (2013);

Fraige etal.

(2019)

3Cannabis

sativa L.

Tetrahydrocannabinol, cannabidiol,

cannabinoids

Analgesic

anticonvulsant,

antidiarrheal, sedative,

relaxant, anxiolytic,

antibacterial, and

antioxidant

Shoot tips, MS medium with

0.3M sucrose, 8g/L agar,

and 5% DMSO

Vitrication Uchendu etal.

(2019); Lowe

etal. (2021)

4Dioscorea

deltoidea wall

Diosgenin Gastrointestinal and

urogenital disorders,

diarrhea, irritability,

abdominal pain,

wounds, intestinal

worms, anemia

Shoot tips, MS medium with

0.3M sucrose, 2M glycerol

and 0.4M sucrose

Vitrication and

the

encapsulation-

dehydration

techniques

Mandal and

Dixit-Sharma

(2007); Dixit etal.

(2005); Semwal

etal. (2021)

M. Silalahi et al.

281

No. Scientic name Secondary metabolites Uses

Types of culture, medium,

and supplements Techniques References

5Gentiana

kurroo Royle

Iridoids, xanthones,

C-glucoxanthone mangiferin, and

C-glucoavones)

Antibacterial,

antioxidant, anti-

arthritic, anti-

inammatory, analgesic

activities and

antidiabetic activity

Shoot tips, Murashige and

Skoog (MS) medium

supplemented with 5%

dimethylsulfoxide (DMSO)

Vitrication and

droplet-

vitrication

Sharma etal.

(2021); Skinder

etal. (2017)

6Kaempferia

galanga L.

Ethyl cinnamate,

P-methoxycinnamate, pentadecane,

δ-selinene, borneol, Eucalypto

Heat cold, dry cough,

toothaches,

rheumatism,

hypertension

Shoot tip MS medium

containing 0.4M sucrose 1.0

mgL−1 BA (6-benzyl

adenine) and 0.5 mgL−1

NAA (α-naphthaleneacetic

acid)

Vitrication Preetha etal.

(2013); Wang

etal. (2021)

7Picrorhiza

kurroa Royle

ex Benth

Glycosides (picroside I, II, and

kutkoside)

Stomachic, purgative

and antiperiodic

treatments, and for

fever and dyspepsia

Shoot tips, medium MS

supplemented with 5%

DMSO

Vitrication Sharma and

Sharma (2003)

8Satureja

spicigera

(K.Koch)

Boiss.

Carvacrol, γ-terpinene, p-cymene Antioxidant Callus MS medium

supplemented with 0.4M

sucrose, 0.5mgL−1 2,4-D,

0.6mgL−1 BAP

Vitrication Ghaffarzadeh-

Namazi etal.

(2017);

Eminagaoglu etal.

(2007)

Biotechnology Toward Medicinal Plants (MPs)

282

7 Conclusion

Biotechnology plays an important role in increasing secondary metabolite/bioactive

compounds and the conservation of MPs. The biotechnologies that can be used to

increase secondary metabolite/bioactive compounds are tissue culture and bioreac-

tor. Meanwhile, the conservation of MPs can be carried out by utilizing invitro

technology, especially invitro cold storage and cryopreservation. This shows that

humans, biotechnology, and plants have a close relationship and can be used to meet

the need for MPs and their conservation. However, the social impact of the use of

biotechnology still needs to be studied.

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Effect of sucrose as an elicitor in increasing quercetin-3-O-rhamnoside (quercitrin) content of chrysanthemum (Chrysanthemum morifolium Ramat) callus culture based on harvest time differences

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Sep 2023

Chrysanthemum (Chrysanthemum morifolium) contains secondary metabolites, such as flavonoid compounds, especially luteolin-7-glucoside and quercetin-3-O-rhamnoside (quercitrin), in its tissues. Utilizing sucrose as an elicitor through callus culture presents an alternative method to enhance the production of secondary metabolites. This research aimed to determine the best sucrose concentration and harvest time for maximizing quercitrin content in chrysanthemum callus culture. The research employed a completely randomized design with four treatment groups: 0, 30, 45, and 60 g/l of sucrose added to MS medium containing 4 ppm 2,4-dichlorophenoxyacetic acid (2,4-D). Callus samples were harvested on the 15th and 30th days of culture. The observed parameters included callus morphology (color and texture), fresh weight, dry weight, the diameter of the callus, and quercitrin content analyzed using high-performance liquid chromatography. The results showed that all callus cultures exhibited intermediate textures and varied colors, predominantly shades of brown. The treatment involving 45 g/l of sucrose with a 30th-day harvest yielded the highest fresh weight, dry weight, and quercitrin content, namely 2.108 g, 0.051 g, and 0.437 mg/g DW, respectively. Notably, the quercitrin content exhibited a 63.67% increase compared to the control.

Optimization of Biomass and Secondary Metabolite Production in Gynura procumbens (Lour.) Merr. Adventitious Roots Culture by Using the Method of Subculture and Fed-batch Cultivation in a Bioreactor

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The valuable extract of bioactive compounds from Gynura procumbens has been widely manufactured into various health products. The demand for these compounds is continuously increasing, but production through conventional farming methods is insufficient due to limited agricultural land and environmental stresses. An alternative to producing plant biomass is in vitro cultivation methods. This method requires less space and enables biomass propagation in a controlled condition that can facilitate stable and efficient production of plant secondary metabolites. This study evaluated the effect of inoculum subculture periods and culture methods on G. procumbens biomass and secondary metabolite production in a bioreactor. The 3-L airlift balloon type-bubble bioreactors was modified in this study to adopt the treatment of 1st-5th subculture periods and fed- and batch-cultivation strategies. We found the G. procumbens adventitious root culture was optimally derived from the 1st subculture produced biomass of 148.02±1.45 g FW and 8.59±0.12 g DW, and TPC (14.48±1.08 mg GAE/g DW) and TFC (116.89±0.44 mg KE/g DW and 33.97±0.13 mg QE/g DW). Additionally, the fed method after 28 days of culture using double distilled water replenishment improved adventitious root biomass (213.75±35.00 g FW and 11.21±0.18 g DW), while nutrient replenishment improved TFC (52.14±0.44 mg KE/g DW and 14.54±0.13 mg QE/g DW). These results can be used to optimize the cultivation of G. procumbens adventitious roots in a large-scale bioreactor.

Production of secondary metabolites using tissue culture-based biotechnological applications

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Jun 2023

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Mar 2023

Sageretia thea is used in the preparation of herbal medicine in China and Korea; this plant is rich in various bioactive compounds, including phenolics and flavonoids. The objective of the current study was to enhance the production of phenolic compounds in plant cell suspension cultures of Sageretia thea. Optimum callus was induced from cotyledon explants on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 0.5 mg L−1), naphthalene acetic acid (NAA, 0.5 mg L−1), kinetin (KN; 0.1 mg L−1) and sucrose (30 g L−1). Browning of callus was successfully avoided by using 200 mg L−1 ascorbic acid in the callus cultures. The elicitor effect of methyl jasmonate (MeJA), salicylic acid (SA), and sodium nitroprusside (SNP) was studied in cell suspension cultures, and the addition of 200 µM MeJA was found suitable for elicitation of phenolic accumulation in the cultured cells. Phenolic and flavonoid content and antioxidant activity were determined using 2,2 Diphenyl 1 picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethybenzothiazoline-6-sulphonic acid (ABTS), ferric reducing antioxidant power (FRAP) assays and results showed that cell cultures possessed highest phenolic and flavonoid content as well as highest DPPH, ABTS, and FRAP activities. Cell suspension cultures were established using 5 L capacity balloon-type bubble bioreactors using 2 L of MS medium 30 g L−1 sucrose and 0.5 mg L−1 2,4-D, 0.5 mg L−1 NAA, and 0.1 mg L−1 KN. The optimum yield of 230.81 g of fresh biomass and 16.48 g of dry biomass was evident after four weeks of cultures. High-pressure liquid chromatography (HPLC) analysis showed the cell biomass produced in bioreactors possessed higher concentrations of catechin hydrate, chlorogenic acid, naringenin, and other phenolic compounds.

New Insights for the Production of Medicinal Plant Materials: Ex Vitro and in Vitro Propagation

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Aug 2023

Medicinal plants are a vital input of raw materials for the manufacture of pharmaceuticals to treat various ailments of humans and animals. The excessive demand for natural medicinal therapies has encouraged intensive harvesting of medicinal plants from the wild reserves without proper replenishment. This has put tremendous pressure on many valuable medicinal plant resources, pushing them toward extinction. Therefore, it is urgent to identify alternative production techniques for medicinal plants to fulfill the growing demand while safeguarding their natural stands. The production of therapeutic herbs has evolved from traditional methods to modern techniques including ex vitro and in vitro propagation and biotechnology. In vitro methods are presented as alternative and complementary techniques of propagation providing solutions for the drawbacks associated with conventional propagation techniques. In addition, to in vitro plant propagation, Synseed production, cell suspension cultures, cryopreservation, short-to-medium-term storage, in vitro metabolite production, and root cultures in vitro are some other important aspects of biotechnological interventions for medicinal plant material production. In this context, studying and distributing knowledge on novel involvements of their production are important to promote collaboration and sharing of information among researchers, scientists, and industry professionals leading to more rapid advancements in the field. Therefore, this chapter highlights the recent knowledge based on medicinal plants giving special reference to ex vitro and in vitro production methods emphasizing the most recent advances, limitations, and future prospects.KeywordsEx vitroIn vitroMedicinal plantsNovel technologiesPropagation

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Jun 2023
FITOTERAPIA

Several plant secondary metabolites are used in the production of different pharmaceuticals based on their biological activities. The conservation and sustainable use of medicinal plants is important for the industrial production of plant-based medicines. Different cryopreservation methods are used for long-term culture preservation, which allows fast regeneration of the preserved plant material with the maintenance of its primary original traits. These methods could ensure the sustainable indefinite supply of plant tissues for theoretically unlimited periods of time, and have gained considerable attention in recent years. It is important to assess the recovery rate and the genetic stability of the recovered plant tissues after cryopreservation because cryopreservation efficiency differs among plant tissues and species. This review lays particular emphasis on the pharmaceutical applications of plant secondary metabolites that are produced through tissue culture approaches, highlighting the methods used for their cryopreservation, as well as their recovery and genetic stability.

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