The investigation of plant-based therapeutic agents in medicinal plants has revealed their presence in the extracts and provides the vision to formulate novel techniques for drug therapy. Vitex negundo (V. negundo), a perennial herb belonging to the Varbanaceae family, is extensively used in conventional medication.

To determine the existence of therapeutic components in leaf and callus extracts from wild V. negundo plants using gas chromatography-mass spectrometry (GC-MS).

In this study, we conducted GC-MS on wild plant leaf extracts and correlated the presence of constituents with those in callus extracts. Various growth regulators such as 6-benzylaminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthylacetic acid (NAA), and di-phenylurea (DPU) were added to plant leaves and in-vitro callus and grown on MS medium.

The results clearly indicated that the addition of BAP (2.0 mg/L), 2,4-D (0.2 mg/mL), DPU (2.0 mg/L) and 2,4-D (0.2 mg/mL) in MS medium resulted in rapid callus development. The plant profile of Vitex extracts by GC-MS analysis showed that 24, 10, and 14 bioactive constituents were detected in the methanolic extract of leaf, green callus and the methanolic extract of white loose callus, respectively.

Octadecadienoic acid, hexadecanoic acid and methyl ester were the major constituents in the leaf and callus methanolic extract. Octadecadienoic acid was the most common constituent in all samples. The maximum concentration of octadecadienoic acid in leaves, green callus and white loose callus was 21.93%, 47.79% and 40.38%, respectively. These findings demonstrate that the concentration of octadecadienoic acid doubles in-vitro compared to in-vivo. In addition to octadecadienoic acid; butyric acid, benzene, 1-methoxy-4-(1-propenyl), dospan, tridecanedialdehyde, methylcyclohexenylbutanol, chlorpyrifos, n-secondary terpene diester, anflunine and other important active compounds were also detected. All these components were only available in callus formed in-vitro. This study showed that the callus contained additional botanical characteristics compared with wild plants. Due to the presence of numerous bioactive compounds, the medical use of Vitex for various diseases has been accepted and the plant is considered an important source of therapeutics for research and development.

India has always been considered a treasure trove rich in medicinal plants and the application of medicinal plants in various forms has been considered a way of life[1]. Eighty percent of the world’s population relies on herbal medicine as primary healthcare. These medicinal plants are an important source of secondary metabolites (SMs), which are small organic molecules that are important for the longevity of plants, but they do not play a role in the growth and development of plants. On the basis of their structures and biosynthesis, these SMs are divided into the following three categories: (1) Phenolic compounds; (2) Terpenoids; and (3) Alkaloids[2]. These SMs are beneficial to human health and effective in preventing diseases. The use of in-vitro propagation techniques facilitates the rapid propagation of rare and commercially important plants. The massive expansion of medicinal plants through the use of plant biotechnology has the potential to meet the pharmaceutical industry’s need for raw materials for herbal preparations. Numerous medicinal plants have been extracted from the natural flora for commercial drug production[3]. The collection of phytochemicals from plant tissues has been studied for over 30 years, and the information obtained contributes to the use of cell cultures to produce the desired phytochemicals. In the last few years, the development of plant tissue culture has increased, and the corporate utility of this technique as a measure of producing precious phytochemicals has received feedback from the scientific community. Cell suspension culture has been found to be the best method for the study of biosynthesis, and callus tissue is the most abundant cell mass obtained during culture production. Cell cultures are useful tools for studying and producing important SMs[4]. Chemical evaluation of plant SMs includes qualitative, quantitative, and biochemical tests. Qualitative and quantitative drug testing can be used to identify the important bioactive components in plant samples.

Vitex negundo (V. negundo), is a small perennial, woody, aromatic and flowering ornamental medicinal herb, commonly known as chaste tree, belonging to the Verbenaceae family. This plant is deciduous in nature and has a medium height of 4-5 m. It has 3 or 5 palmate-sized flowers, with deep-violet purple aromatic flowers and rounded black-ripe fruit. Plants of Vitex species have different ethno-botanical and pharmacological applications. V. negundo contains many bioactive phytochemicals such as glycosides, phenolic compounds, flavonoids, terpenes and phytosteroids. Terpenoids are thought to be one of the most abundant metabolites in the Vitex plant. Chemically, they contain five carbon isoprene units, forming monoterpenes (five carbon atoms), hemiterpenes (C5), sesquiterpenes (fifteen carbon atoms), diterpenes (twenty carbon atoms), and triterpenes (thirty carbon atoms). The leaves of Vitex are used to reduce breast tenderness, regulate hormones related to fertility, menstrual cycle, menstrual pain and amenorrhea symptoms. Pharmacologically, this species showed anti-oxidative, antimicrobial, anti-inflammatory and anti-tumor properties. It reduces the level of serum prolactin in hyper-prolactinemia and mastodynia. The leaves of V. negundo are aromatic, bitter, pungent, and have astringent, analgesic, anti-inflammatory, antipyretic, and anthelmintic properties. Literature has revealed that leaves and flowers of Vitex are the main sources of SMs. Leaves are a good source of alkaloids, vitamin C, carotene, glycol-nonanol, benzoic acid, β-sitosterol, flavonoids (such as luteolin-7-glycosides, ricin, iridoid glycosides, C-glycosides), terpenes oil (such as caryophyllene epoxide, δ-guaiene, and ethyl-hexadecenoate), while flowers contain oil such as α-selinene, (E)-nerolidol, carryophyllene epoxide, and germacren-4-ol. To date, there is no comprehensive record available on the phytochemical study of wild leaf and callus culture (in-vitro) samples obtained from this plant. Thus, to determine the therapeutic importance of V. negundo, a detailed comparative study of phytochemical constituents is essential. Hence, the aim of this study was to analyze and identify the phyto-constituents of wild leaves and in-vitro cultured leaf calluses and to spectroscopically identify bioactive compounds in crude extracts prepared in methanol by gas chromatography-mass spectrometry (GC-MS) analysis.

The GC-MS chromatogram spectra were achieved in all three extracts of V. negundo i.e., wild in-vivo leaves, green-compact and white-loose in-vitro callus. The results showed that bioactive compounds were present in both in-vivo and in-vitro sample extracts (Figure 1). Using GC-MS analysis we detected twenty-four, ten, and fourteen bioactive compounds in the methanolic extract of wild-leaves, green, and white loose callus, respectively (Tables 2-4). The results showed that octadecadienoic acid, hexadecanoic acid and methyl esters were the main components in the methanol extract of leaves and callus. Among these, octadecadienoic acid was the most common compound in all samples[7,8]. The maximum concentration of octadecadienoic acid in leaves, green callus and white loose callus was 21.93%, 47.79% and 40.38%, respectively. Our results confirmed that the concentration of octadecadienoic acid doubled in-vitro compared to in-vivo. In earlier research, the results of GC-MS analysis revealed similar compounds to those obtained in the present study[9].

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Figure 1 Gas chromatography-mass spectrometry chromatogram of the methanol extract of wild leaves, methanol extract of green callus, and methanol extract of white loose callus of Vitex negundo (L.). A: Methanol extract of wild leaves; B: Methanol extract of green callus; C: Methanol extract of white loose callus.

In V. negundo plants, octadecadienoic acid is present along with plant glycosides[10]. Plant glycosides are a diverse group of natural compounds found in various parts of plants, including leaves, stems, roots, and seeds. They consist of a sugar molecule (glycone) attached to a non-sugar, biologically active compound (aglycone or genin), which has both medicinal and toxic properties[11]. Along with the plants’ glycosides, octadecadienoic acid develops bitterness in leaves, which frightens herbivores as the leaves are toxic or unpleasant when ingested[12]. Some important glycosides, e.g., anthocyanin glycosides, along with octadecadienoic acid are responsible for the pigmentation (red, blue, and purple) in leaves, which act as stressors in plant defense against temperature stress[13].

Other bioactive constituents such as 1-oxo-dimethyl-methylene-hexahydrocyclopentanol pyran (7.91%), veridiflorol (6.79%), pyrrolo-carbazole (CAS) (6.79%), and dimethyl-phenyl (6.79% peak area) were identified in the methanolic leaf extract. It was observed that other bioactive compounds such as dursban, butyric acid, benzene, 1-methoxy-4-(1-propenyl), nor-ses-terterpene-diester, tri-decanedial, chlorpyrifos, methyl-cyclohexenyl-butanol, and anhalonine were present in good amount in in-vitro developed callus (both green-compact and white-loose) along with the major compounds (octadecanoic acid, hexadecanoic acid, and methyl ester) (Tables 3 and 4). The results obtained in the present study were analogous with the data obtained by Kaliyannagounder et al[9]. In medicinal plants, octadecadienoic acid is an important polyunsaturated fatty acid. It is also known as linoleic acid and plays several important roles in plant growth and development. Octadecadienoic acid triggers the production of defensive compounds in plants and plays a major role in plant defense mechanisms. It is considered a core-structural integral element of phospholipids that make up the lipid bi-layer of plant cell membranes. It maintains the integrity of the plant cell and controls the movement of molecules in and out of the cell along with other fatty acids. Finally, it adjusts the fluidity and stability of membranes under different environmental stresses, such as temperature extremes, drought, and pathogen attacks. Furthermore, it acts as a precursor of jasmonic acid, which is a signaling molecule involved in the plant’s response to herbivores, pathogens, and other stressors in plant defense mechanisms. These findings are similar to those obtained by Ahuja et al[14].

Hexadecanoic acid and methyl esters have antioxidant, cholesterol-lowering, antiandrogenic, hemolytic, and alpha-reductase inhibitory properties. Hexadecanoic acid (palmitic acid) is a common saturated fatty acid, while hexadecanoic acid methyl ester (methyl palmitate) is a chemically modified derivative of palmitic acid. On the basis of data available in the literature, plant biomass with good amounts of methyl palmitate (a chemically modified derivative of hexadecanoic acid) is used in biodiesel production. The present study found that in-vitro grown callus (white-loose) is a good medium for the synthesis of methyl esters, and we can consider this approach in the near future as a source of renewable fuel for our future energy demands, if appropriate research is carried out[15].

In the present study, two major compounds were found i.e., a sesquiterpenoid compound “viridiflorol” and anhalonine (naturally occurring alkaloid) in the in-vitro-derived white loose callus extract of V. negundo[16,17]. Viridiflorol showed anti-inflammatory, antioxidant, and anti-mycobacterium tuberculosis activity. Anhalonine is considered an important naturally occurring alkaloid. A very small amount of this compound was obtained from Lophophora williamsii (a rare species of cactus). It may serve as a chemical defense mechanism in some plants, and may be toxic to herbivores and pathogens. Hence, by producing anhalonine, plants may discourage herbivores and reduce the risk of damage from grazing animals. It seems that anhalonine also showed allelopathy. Such compounds are released by the plants into the soil through their root system and inhibit the growth of nearby competing plants and provide a competitive advantage for the producing plant. It was noted in the present work that plants of Vitex grown in the in-vivo field condition usually inhibited the growth of weeds in their nearby area. This may be due to the presence of anhalonine, which was not detected in the methanolic extract of the wild leaf sample. The compound was present in very good amounts (peak area 1.43%) in white loose callus. Anhalonin, used as a psychotropic drug, can change the function of the nervous system and results in alterations of perception, mood, cognition, and behavior in our traditional folk medicinal/herbal system. It also showed antimicrobial properties, which protect the plants from microbial infections and diseases by acting as natural antimicrobial agent[18]. The exact role of anhalonine in plants has not been extensively studied. Detailed research on the functions of anhalonine in different plant species is continuing, and further studies are required to clarify its ecological and physiological significance in the plant system.

Similar outcomes were obtained in a previous study conducted by Lad et al[19]. The methanolic extract of V. negundo showed the presence of phytol compounds, which have other biological effects such as hypocholesterolemic cancer prevention, insecticidal, hepatoprotective, inhibition of 5-alpha reductase, anti-inflammatory, anti-rash, nematocidal, antihistamine, anti-acne, and anti-inflammatory antibiotic properties. The same bioactive SM of phytol has been previously reported to have various medicinal properties in some aquatic plant species such as Hydrilla verticillata, Gracilaria, and Carissa carandas[20,21]. Octadecanoic acid is a well-known example of a saturated fatty acid and possesses antihypertensive properties along with the ability to decrease low density lipoprotein cholesterol and increase high density lipoprotein cholesterol levels[22].

In the present study, octadecanoic acid was present in both leaf and callus extracts of V. negundo. The results of the present study were in accordance with reports on Cleistanthus collinus, Goniothalamus umbrosus, Kigelia pinnata, and Melissa officinalis which contained n-hexadecanoic and octadecadienoic acids[23,24]. The study revealed the presence of various bioactive compounds present in all the methanolic extracts of in-vivo and in-vitro plant samples[25]. However, it has become clear from the present study that the callus contained additional phytochemicals in comparison to wild-type plants that display significant medicinal properties. Further studies such as bio-prospecting are necessary to support the biological properties and the importance of these inventive bio-molecules.

Screening of the methanolic extracts of Vitex negundo revealed the presence of twenty-four, ten, and fourteen bioactive compounds in the wild-leaves, green, and white loose callus, respectively. The results confirmed that octadecadienoic acid, hexadecanoic acid, and methyl ester were found to be the key constituents in the methanolic extract of leaves and callus[26]. Octadecadienoic acid was the predominant potential bioactive compound identified in all samples. Results of the present research confirmed that the concentration of octadecadienoic acid in in-vitro conditions was twice that in in-vivo conditions. Hence, in the case of Vitex, we can use micro-propagated plants as a potent source of phyto-compounds for commercialization without destroying the wild plant population[27,28]. A recent literature review showed that plant biomass with good amounts of methyl palmitate (a chemically modified derivative of hexadecanoic acid) is used in biodiesel production[29,30]. The findings of the present study showed that in-vitro grown callus is a good medium for the synthesis of methyl esters. Hence, we can consider this approach in the near future as a source renewable fuel for our future energy demands. Active sesquiterpenoid compounds such as viridiflorol and anhalonine (naturally occurring alkaloid) were found in the in-vitro derived white loose callus extract of Vitex negundo. Viridiflorol showed anti-inflammatory, antioxidant, and anti-mycobacterium tuberculosis activity, while anhalonine may be used as a psychotropic drug in animals and is also responsible for showing allelopathy in plants. This study showed that the callus contained additional botanical characteristics in comparison to wild plants. Due to the presence of numerous bioactive compounds, the medical use of Vitex callus for various diseases has been accepted and the plant is considered an important source of therapeutics for research and development.

Authors are grateful to Dr. Shoor Vir Singh, Professor & Head, Department of Biotechnology at GLA University, Mathura for help and support during the present study.