INTRODUCTION:

Endangered medicinal plants i.e. Rauwolfia serpentina, Adhatoda vasica and Alstonia scholaris belong to Apocynaceae family. Apocynaceae is a family of flowering plants that includes herbs, shrubs, trees and vines, commonly known as dogbane family, because some taxa were used as dog poison. Members of family Apocynaceae are rich in alkaloids, terpenoids, steroids, flavonoids, glycosides, simple phenols, lactones, and hydrocarbons. Few studies have reported antioxidant, anti‐inflammatory, antimicrobial, and cytotoxic activities of crude extracts as well as single compounds isolated from various members of the family Apocynaceae¹.

Species of Apocynaceae are consumed by people in the rural area as a food and some others are used as a poison². The plants of family Apocynaceae are native throughout India, Pakistan, China, Bangladesh, and Sri Lanka³. Majority of the medicinal plants and metabolites were obtained from this family. Medicinal plants are known to harness a number of bio active compounds with potent pharmacological significance. Many plant species have been reported to possess antimicrobial activities⁴.

This species does not have a local name, so there is no local knowledge regarding its potential⁵. In Andhra Pradesh, India, Rauwolfia serpentina, Adhatoda vasica and Alstonia scholaris included in the list of medicinal plants with the International Union for Conservation of Nature (IUCN) status as endangered or threatened species⁶. The effective protocol micro propagation is a successful means for rapid multiplication of endanged and endemic species by using conventional methods. These methods have some limitations to regenerate and development of invitro strategies for conservation. Micropropagation not only ensures the supply of quality planting material and also storage of germplasm in vitro cultures as an additional advantage^7,8.

Maximum alkaloids, yohimbine, reserpine, serpentine, ajmalicine deserpidine and ajmaline are used to treat hypertension and breast cancer. The crude extract including Reserpine, used as a natural tranquilizer was found to be hypotensive activity^9,10. In general Rauwolfia serpentina roots are generally obtained through shoot organogenesis and callus morphogenesis^11,12.

Limited literature on these important endemic medicinal plants i.e. Rauwolfia serpentina, Adhatoda vasica and Alstonia scholaris collected from Eastern Ghats of Andhra Pradesh. This is the first report on estimation of Phytochemical, antimicrobial and micro propagation of these medicinal endemic plants. In this paper, we report the Antimicrobial, Micropropagation, Phyto chemical estimation and quantitative estimation of Alkaloids, phenols, flavonoid and Terpenoids from Rauwolfia serpentina, Adhatoda vasica and Alstonia scholaris.

MATERIALS AND METHODS:

Plant Material:

Endemic medicinal plants Rauwolfia serpentina, Adhatoda vasica and Alstonia scholaris were collected from higher altitudes of Easter Ghats, Lambasingi forest region, Visakhapatnam, Andhra Pradesh. The collected plant materials were grown in pots under suitable climatic condition and this material was used for in-vitro studies i.e. in micro propagation.

Preparation of extracts:

The plant parts were collected and shade dried for about two weeks and ground into coarse powder. About 100g powder extracted with 500ml of petroleum ether using soxhlet apparatus. The same plant powders were also extracted with chloroform, acetone and methanol. The extracts were concentrated to dryness to yield crude residue. These residues were used for preliminary phytochemical analysis¹³.

Quantitative estimation of phytochemical constituents:

Chemicals and reagents:

From SD Fine Chemicals, Mumbai, Agar-Agar and α-Naphthalene Acetic Acid (NAA) were purchased. 6-benzyl aminopurine (BAP), 3-indolebutyric acid (IBA), Indole-3-acetic acid (IAA), Murashige and Skoog (MS) media components were purchased from Merck Ltd. Mumbai; and from Sisco Research Ltd. Mumbai, paraamino benzoic acid was delivered.

Antibacterial assay:

By using agar well diffusion method, the antimicrobial activities of methanolic extracts of plant like leaves, follicles and latex were analysed against Aspergillus species and Pseudomonas species. Nutrient Agar medium was prepared and sterilized in an autoclave at 121°C at 15 lb pressure for exact 15 minutes. For the preparation of culture plates, 20ml of the sterilized medium was poured in petri plates or dishes. After solidification, with the help of sterilized L or V shaped glass rod 1ml of the inoculum suspension was uniformly spread over the agar in perti dishes. In each plate 6 mm wells were made with sterile cork borer. The wells were loaded aseptically with extracts 100µl (50,100mg/ml concentration) which was dissolved in dimethyl sulfoxide (DMSO) solution. At the same time, a control with DMSO was also maintained. In the petri dishes, the microbial growth was observed after the incubation period. The mean of diameter of the inhibition expressed, was considered as the antimicrobial activity.

Sterilization of explant:

Under running tap water the explants were placed for 20 min and then washed with a mixture of 2 drops of Dettol and 1 drop of Tween 80 in 150ml of distilled water for nearly 10 min then they are thoroughly washed with distilled water for the elimination of traces of germicidal agents (Pandey et al., 2010). With 0.1%w/v mercuric chloride (MgCl₂), they were surface-sterilized for 10 min and then washed five times with sterile distilled water. Small pieces of disinfected explants (1.5-2.0 cm) were placed aseptically onto MS medium with pH 5.6, adjustments were made prior to sterilization in an autoclave at 121°C and 1.06kg/cm² for 20 min), supplemented with growth regulators, 3% sucrose and 0.6% agar. And then the cultures were incubated at 25 ± 2°C using white fluorescent lamps with a light intensity of 3000-Lux. A photoperiod of 16/8 light and dark cycles was also maintained.

Shoot induction:

Surface sterilized explants were cut to 1cm length, each with a single node. Then they were inoculated in a vertical manner on the surface of culture medium. Then the cultures were grown under 16 h photoperiod with a light intensity of 2000-3000 lux, at 25±1°C. In vitro shoot induction of Toddalia asiatica nodal explants on MS medium supplemented with various concentrations of BAP at different concentrations were maintained.

Root induction:

For root induction, well grown shoots were inoculated on half strength of MS medium with different concentrations and different combinations of IBA and NAA.

Statistical Analysis:

All measurements were carried out in triplicate. Statistical analyses were performed using one-way analysis of variance (ANOVA), and the significance of the difference between means was determined by Duncan’s multiple range tests. Differences at 𝑃 < 0.05 were considered statistically significant.

RESULTS AND DISCUSSION:

The phyto chemical screening of the selected plant species i.e. Rauwolfia serpentina, Adhatoda vasica and Alstonia scholaris were revealed the primary and secondary metabolites. Rauwolfia serpentina plant extract shows the metabolites like amino acids, alkaloids, carbohydrates, flavonoids, phenols, terpinoids, oils, Saponins, sterols and lipids. The other two medicinal plants Alstonia scholaris and Adhatoda vasica also showed the majority of these metabolites. Table-1 represented the strong (++), moderate (+) and weak (-) metabolite production by these medicinal plants. Lipids were not observed in Rauwolfia serpentina and Alstonia scholaris. Glycerides were not observed in these three plants.

The quantitative estimation revealed that the plant extract exhibited alkaloids, flavonoids, Saponins and amino acids. Rauwolfia serpentina plant extracts in the presence of methanol showed the maximum Saponins (57.15±1.2) followed by flavonoids (35.02±0.5), alkaloids (29.05±0.7) and amino acids (24.16±0.5). In the present study Saponins were identified as maximum content was produced by all these three important medicinal plants.

Antimicrobial activity of the extract from adult plant and in vitro calli was determined by agar-diffusion assay with test bacteria spread on Mueller Hinton Agar plates and fungi spread on Sabouraud’s Dextrose Agar plated by¹⁴. The researcher Britto et al. stated on other apocynacea members like Rauvolfia tetraphylla, Nerium oleander, Tabernaemontana divaricate, and Allamanda cathartica, for phytochemical analysis and antibacterial activity¹⁵. Secondary metabolites like Sterols, tannins and saponins from Apocynaceae members are reported to be responsible for antimicrobial activity was reported¹⁶.

Table-1: Phytochemical screening of selected medicinal plants

Phytochemical tests	*Rauwolfia serpentina*			*Alstonia scholaris*			*Adhatoda vasica*
Phytochemical tests	E.A.	Methanol	Chloro form	E.A.	Methanol	Chloro form	E.A.	Methanol	Chloro form
Amino acids	++	++	++	++	++	++	++	++	++
Alkaloids	++	++	++	++	++	++	++	++	++
Carbohydrates	+	+	-	+	-	+	-	+	-
Tannins	-	-	-	-	-	-	+	+	+
Flavonoids	++	++	++	++	++	++	++	++	++
Phenols	+	+	+	+	+	+	-	-	-
Terpenoids	+	+	+	-	-	-	+	+	+
Oils	+	+	+	+	+	+	+	+	+
Saponins	++	++	++	++	++	++	++	++	++
Glycosides	-	-	-	-	-	-	-	-	-
Sterols	+	+	+	-	-	-	+	+	+
Lipids	-	-	-	-	-	-	+	+	+

(+= normal reaction, ++= strong reaction)

Table -2 Quantitative estimation of extracts from Rauwolfia serpentina

Sl No	Name of the extract	Total alkaloid content	Total Flavonoids content	Total Saponins content	Total amino acids content
1	Ethyl acetate	22.03±1.3	21.23±0.6	35.05±0.1	15.05±0.2
2	Chloroform	18.55±0.2	19.08±0.7	28.05±0.4	21.07±1.5
3	Methanol	29.05±0.7	35.02±0.5	57.15±1.2*	24.16±0.5

Means followed by *in column are significantly different at p≤0.05

Table -3 Quantitative estimation of extracts from Alstonia scholaris

Sl No	Name of the extract	Total alkaloid content	Total Flavonoids content	Total Saponins content	Total amino acids content
1	Ethyl acetate	22.08±2.3	19.05±0.7	13.06±1.2	11.06±3.2
2	Chloroform	28.05±1.1	23.05±1.3	22.09±0.1	16.05±0.5
3	Methanol	36.05±1.4	30.04±1.2	55.06±0.7*	18.14±0.5

Means followed by *in column are significantly different at p≤0.05

Table -4: Quantitative estimation of extracts from Adhatoda vasica

Sl no	Name of the extract	Total alkaloid content	Total Flavonoids content	Total Saponins content	Total amino acids content
1	Ethyl acetate	19.03±1.0	25.05±0.5	25.07±0.3	18.06±0.1
2	Chloroform	26.06±0.4	22.05±0.9	21.06±0.2	22.06±0.5
3	Methanol	32.06±0.8	42.05±0.7	49.16±1.5*	28.29±0.4

Means followed by *in column are significantly different at p≤0.05

Table-5: Zone of inhibition on antimicrobial activity of compounds extracted from aerial parts of the Rauwolfia serpentina

Extract (200 mg/ml)	Zone of inhibition (mm)
	*S. pyogenes*		*P. aeruginosa*		*Aspergillus niger*		*V. cholera*
	5%	10%	5%	10%	5%	10%	5%	10%
Ethyla Acetate	9.3±0.0	11.5±0.09	7.5±0.5	10.3±0.5	8.3±0.4	9.3±0.33	8.8±0.5	14±0.0
Chloroform	10.0±0.33	13.5±0.3	9.1±0.45	13.5±0.45	10.0±0.33	13.3±0.9	8.5±0.81	15.5±0.5
Methanol	11.5±0.5	19.5±0.5	9.8±0.5	14.0±0.5	13.8±0.8	20.0±0.0	10.2±0.3	17.8±0.45
Chloramfenicol (10µg/ml)	75±0.5	75±0.5	40±0.5	40±0.8	65±0.5	65±0.7	-	-

(-- No antimicrobial activity)

Figure-1: Application of plant growth regulators in various combinations for Rauwolfia nodal segments

The selected plant extracts were showed the antibacterial and antifungal activity against the organisms (Table-5). The results revealed that methanolic extracts exhibited considerable zone of inhibition against S. pyrogens, P. aeruginosa, A. niger and V.cholera. The ethyl acetate and chloroform extract has showed moderate activity against both the organisms at 20% concentration. All the extract was compared with the standard drug Chloramphenicol. All the extracts exhibited antibacterial activity at different concentrations (5% and 10%). Anti bacterial activity and Anti fungal activity the present strains showed maximum zone of inhibition at all the concentrations. All the extracts were showed comparable activity with the standard drug Chloramphenicol.

From the results maximum (95%) callus induction response was observed on 15^th day of inoculation from nodal explants under dark conditions on MS medium supplemented with 6BAP (1.0mg/L) + KIN (1.0mg/L). For shoot and root proliferation, MS basal medium (1.0, 2.0, 3.0 and 4.0%) supplemented with 6 BAP, KIN and TDZ. MS medium with 6BAP combination was gave best rooting response with quality roots (Figure-1). In the study, we were selected Rauwolfia for the nodal segments were cultured by using different plant growth regulators individually and in combination, in order to roll out the most efficient PGR individually or in combination, to propagate the Rauwolfia serpentina. Plant growth regulators (PGRs) employed were 6‐ Benzylaminopurine (BAP), kinetin, 1‐Naphthaleneacetic acid (NAA), Thidiazuron (TDZ), and adenine sulphate for multiple shoot induction from nodal cuttings of the wild plant. Similar attempts have been made for the propagation of Carissa carandas using shoot apex and nodal part^17,18.

CONCLUSION:

Alkaloids, terpinoids, flavonoids and phenols are reported for the first time in Rauwolfia serpentina, Adhatoda vasica and Alstonia scholaris plants. Methanolic extract Rauwolfia serpentina, Adhatoda vasica and Alstonia scholaris exhibited alkaloids, flavonoids, amino acids and Saponins. In this study for the first time we are reported maximum Saponins were defined. Methanol extraction of Rauwolfia serpentina showed antimicrobial activity against Vibrio cholera along with other bacteria tested. Micro propagation of this plant is very high like 95%.

ACKNOWLEDGEMENT:

Author would like to thank the department of Botany and Andhra Christian College for providing necessary facilities to complete this research work.

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Received on 02.09.2020 Modified on 10.12.2020

Asian Journal of Pharmacy and Technology. 2021; 11(2):111-115.

DOI: 10.52711/2231-5713.2021.00018