Evaluation of phytochemical, antioxidant and antimicrobial...

Indian Journal of Natural Products and Resources,

Vol. 4(4), December 2013, pp. 363-370

Evaluation of phytochemical, antioxidant and antimicrobial activities of

Memecylon L. species from Western Ghats

A R Sivu1, #

, N S Pradeep1*, K B Rameshkumar

2 and A G Pandurangan

1

1Plant Systematic and Evolutionary Science Division, 2Phytochemistry and Phytopharmacology Division

Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram-695 562, Kerala, India

Received 18 April 2012; Accepted 29 July 2013

The genus Memecylon L. is a least explored group of plants and the present study reports the preliminary phytochemical

and pharmacological evaluation of 32 Memecylon species collected from the Western Ghats. Among these 4 are rare and

endangered and 16 endemic. The methanolic extracts of the leaves were evaluated for their phenolic and flavonoid contents,

in vitro antioxidant and antimicrobial activities. Phenolic contents of the methanol extracts of leaves were comparatively

low in the species, ranging from 89.86 mg/g (M. gracile Bedd.) to 05.04 mg/g (M. depressum Benth.). Flavonoid compounds

were highest in M. grande Retz. (39.56 mg/g) and lowest in M. talboltianum Brandis (07.60 mg/g). The in vitro free radical

scavenging assay was done using DPPH method and the results showed moderate activities to the leaf extracts. The highest

antioxidant activity was observed for M. heyneanum Benth. (IC50=15.30 µg/mL). Antibacterial activity against three Gram-

positive and six Gram- negative bacteria was tested by disc diffusion method and the leaf extracts showed remarkable broad

spectrum antibacterial activity, especially for M. deccanense Clarke, M. terminale Dalzell, M. heyneanum Benth.,

M. clarkeanum Cogn. and M. sessile Benth.

Keywords: Memecylon species, Western Ghats, Phenolics, Flavonoids, Antioxidant, Antibacterial.

IPC code; Int. cl. (2011.01)−A61K 36/00.

Introduction India has a rich floristic diversity with 46, 550

plant species that accounts for about 11% of the total

world flora, of which 28 % are endemic to the

country1. Among the four hot spots of floristic

diversity in India, the Western Ghats hosts nearly

4500 flowering plants are endowed with high

endemism with nearly 1500 endemics. Most of

the floristic wealth, especially the endemic species

remains unexplored regarding their potential utility

and the chemical constituents or bioactivities.

The wild underutilised plants have become an

interesting source of nutraceutical, healthcare,

cosmetic and food industries. Indiscriminate

exploitation, habitat destruction and introduction

of exotic species lead to the reduction of population

of the endemic, rare and endangered species and

therefore there is an urgent need to conserve these

species along with evaluation of their potentialities.

The genus Memecylon L. is among the least explored

group of plants and hence an attempt has been made

to evaluate their phytochemical and pharmacological

potential.

The genus Memecylon L., belonging to the

family Melastomataceae, is represented world over

by around 250 species of shrubs and trees in the

paleotropical region3. They are distributed in all

types of habitats ranging from deciduous, semi

evergreen, evergreen and montane forests with a wide

range of altitude from sea level to 2000 m. In India

the genus Memecylon is represented by 39 species of

which 21 are endemic to the country and the Western

Ghats is reported to host 29 species4-8

. However,

while studying the molecular taxonomy of the genus

Memecylon L. of the Western Ghats, the authors did

collect 3 more Memecylon species (M. procerum Thw.,

M. clarkeanum Cogn. and M. parvifolium Thw.) as

new records to India (Plate 1). Among 32 species

collected from the Western Ghats, 4 species, viz.

M. lawsonii Gamble, M. lushingtonii Gamble,

M. flavescens Gamble and M. sisparense Gamble

comes under rare and endangered categories (Plate 1),

while 16 species are endemic to the region9 (Table 1).

——————

*Correspondent author:

Phone: 0472 2869226

E-mail: [email protected] #

Present Address: Department of Botany, NSS College Nilamel,

Kollam-691 535, Kerala

INDIAN J NAT PROD RESOUR, DECEMBER 2013

364

Plate 1—Common Memecylon species (a: M. umbellatum, b: M. edule and c: M. randerianum); Rare and endangered

species (d: M. sisparense, e: M. lawsonii, f: M. flavascence and g: M. lushingtonii); New records to India (h: M. parvifolium, i:

M. procerum and j: M. clarkeanum)

SIVU et al.: PHYTOCHEMICAL, ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF MEMECYLON L. SPECIES

365

Memecylon species are utilized world wide as

timbers, ornamentals, source of edible fruits and

yellow dye in addition to their medicinal properties3.

Among the different Memecylon species, M. umbellatum

Burm., is the most common species (Plate 1), distributed

in India and Sri Lanka and known as ‘iron wood tree’

(locally known as Kasavu, Kayampoo, Kanalei). The

plant is popular as ornamental tree and widely used in

traditional medicines and also as source of food, dye

and timber. The pulpy, astringent ripe berries of the

tree are edible and are eaten in time of famine.

The leaves yield a yellow dye and in combination

Table 1—Total phenolic, flavonoid compounds and DPPH scavenging activity of the leaf methanol extracts of Memecylon species

M. sp.

ID

Memecylon species Voucher

No

Total

Phenolics

Total

Flavonoids

IC50 values

(µg/mL)

M 1 M. agastyamalaianum Santhosh et al * 65102 38.03 ± 0.02 08.40 ± 0.04 87.01 ± 0.01

M 2 M. angustifolium Wight 65106 23.97 ± 0.02 10.03 ± 0.04 87.08 ± 0.02

M 3 M. capitellatum L. 63524 06.04 ± 0.02 31.93 ± 0.04 115.37 ± 0.04

M 4 M. depressum Benth. * 61748 05.04 ± 0.01 17.76 ± 0.03 128.13 ± 0.03

M 5 M. flavescens Gamble* 65126 05.05 ± 0.01 11.23 ± 0.04 115.17 ± 0.04

M 6 M. gracile Bedd. * 65104 89.86 ± 0.04 08.42 ± 0.01 58.56 ± 0.04

M 7 M. grande Retz. 61736 09.90 ± 0.04 39.56 ± 0.04 64.00 ± 0.02

M 8 M. heyneanum Benth. 63543 61.06 ± 0.03 12.40 ± 0.04 15.30 ± 0.03

M 9 M. clarkeanum Cogn. 63600 59.96 ± 0.04 11.30 ± 0.03 97.37 ± 0.01

M 10 M. lushingtonii Gamble 65146 10.13 ± 0.03 16.70 ± 0.03 148.23 ± 0.04

M 11 M. malabaricum (C. B. Clarke.) Cogn. 63518 25.76 ± 0.03 12.56 ± 0.05 207.24 ± 0.03

M 12 M. rivulare Bremer 61767 15.83 ± 0.04 13.63 ± 0.03 141.20 ± 0.02

M 13 M. sessile Benth. 65120 82.83 ± 0.03 09.16 ± 0.04 40.10 ± 0.04

M 14 M. sisparense Gamble* 65124 30.36 ± 0.06 09.13 ± 0.04 44.06 ± 0.02

M 15 M. sivadasanii Mohanan et al * 61775 14.06 ± 0.01 10.80 ± 0.02 55.10 ± 0.03

M 16 M. subramanii Henry* 63564 09.46 ± 0.04 14.96 ± 0.04 91.06 ± 0.03

M 17 M. talboltianum Brandis* 65161 11.03 ± 0.05 07.60 ± 0.03 114.0 ± 0.02

M 18 M. terminale Dalzell* 65162 65.96 ± 0.15 10.60 ± 0.02 232.47 ± 0.03

M 19 M. umbellatum Burm. 61751 10.10 ± 0.03 15.33 ± 0.05 62.43 ± 0.04

M 20 M. wightii Thw. 63584 06.78 ± 0.03 09.56 ± 0.04 77.21 ± 0.02

M 21 M. mundanthuraianum Viswanathan & Manik.* 65116 51.76 ± 0.04 07.53 ± 0.03 214.27 ± 0.05

M 22 M. bremeri Viswanathan 65136 10.75 ± 0.03 20.33 ± 0.04 53.90 ± 0.03

M 23 M. manickamii Murugan et al * 65171 14.10 ± 0.03 12.50 ± 0.04 85.37 ± 0.03

M 24 M. courtalensis Manickam et al * 63540 05.63 ± 0.05 09.00 ± 0.02 74.22 ± 0.02

M 25 M. tirunelvelicum Murugan et al * 65121 23.73 ± 0.05 13.53 ± 0.04 149.97 ± 0.04

M 26 M. royenii Blume 65138 07.83 ± 0.03 12.50 ± 0.04 145.17 ± 0.04

M 27 M. deccanense Clarke* 65165 67.06 ± 0.01 08.46 ± 0.04 110.00 ± 0.05

M 28 M. edule Roxb. 63558 08.73 ± 0.04 14.36 ± 0.03 82.43 ± 0.03

M 29 M. parvifolium Thw. 63563 64.10 ± 0.03 10.40 ± 0.05 162.37 ± 0.05

M 30 M. procerum Thw. 61752 05.90 ± 0.10 18.20 ± 0.03 103.97 ± 0.03

M 31 M. lawsonii Gamble* 61761 08.83 ± 0.03 12.43 ± 0.04 114.17 ± 0.02

M 32 M. sylvaticum Thw. 61726 15.86 ± 0.02 15.30 ± 0.05 78.13 ± 0.02

* Endemic to the Western Ghats. Phenolic contents: mg gallic acid equivalent per g dry weight of leaves methanol extract. Flavonoid

contents: mg quercetin equivalent per g dry weight of leaves methanol extract. Values are mean ± SD of triplicate experiments.


366

with myrobalans and sappan wood it produces

bright red tinge10

. In traditional medicines, leaves of

M. umbellatum has been used as a cooling astringent,

in conjunctivitis as a lotion and given internally in

leucorrhoea and gonorrhoea11

. The leaves are also

used for the treatment of diabetes12

, in the treatment

of bone fracture13

, herpes14

, skin diseases15

and snake

bite16

. The leaves and barks are applied to bruises10

.

The decoction of the root is used in excessive

menstrual discharge17,18

. The seeds are used to cure

cough and are sedative19

. Biological activities like

anti-diabetic20-21

, antibacterial22

, antiviral23

and wound

healing activities24

of M. umbellatum leaves have

been reported. The roots also possess analgesic25

and

hepatoprotective26

activities.

Memecylon edule Roxb. is another common

species (Plate 1), well known for its use as a mordant

in silk dyeing. Treatment with the leaf of M. edule is

said to heal burning wounds without any scar. The

anti-inflammatory, analgesic and antioxidant activities

of the leaves supported the traditional use of the

leaves in relieving inflammation and pain27

.

Decoction of roots and heartwood has also been used

to relief fever symptoms of several diseases such as

common cold, measles and chicken-pox27

. The

antibacterial activity of the seed extract of M. edule

has also been evaluated28

. M. malabaricum Cogn.

(syn. M. randerianum Almeida & Almeida).

is a common species (Plate 1) and decoction

of the flowers and twigs is used to treat skin diseases

and chicken pox10

. A paste of young shoot tips

and cumin seeds are applied against herpes and

the decoction of young leaves in combination is

taken against stomach disorders29

. The leaves possess

antimicrobial activity30-31

. The leaves of M. lushingtonii

Gamble has been reported as an effective post-coital

contraceptive32

. The bark of M. angustifolium Wight is

used as a tonic and refrigerant18

.

Out of the 32 Memecylon species collected from

the Western Ghats region, literature survey shows that

only M. umbellatum has been studied for their

chemical constituents. Preliminary phytochemical

screening revealed the presence of phenolic

compounds, flavonoids, steroids and terpenoids in

different extracts of the leaves of M. umbellatum22

.

The proximate components, nutritive values and

elemental analysis of the leaves have been reported33

.

Tannins have been reported in significant quantity

in the bark, followed by leaves, roots, stem and

inflorescence of M. umbellatum34

. α-Amyrin, sitosterol,

oleanolic acid, ursolic acid, sitosterol-β-D-glucoside

and umbelactone have been isolated from the aerial

parts of M. umbellatum Burm.35

. The roots yielded,

octocosonoic acid, cerotic acid, ethyl palmitate,

palmitic acid and butyric acid36

.

The present study reports the quantitative estimation

of the phenolic compounds and flavonoids of the

methanol extracts of the leaves of 32 Memecylon

species distributed in the Western Ghats region of

India along with their in vitro free radical scavenging

activities and antimicrobial activities.

Materials and Methods

Plant Material

Fresh leaves of the Memecylon species (Table 1)

were collected from different localities, including type

localities in the Western Ghats region and voucher

herbarium specimens were deposited in Jawaharlal

Nehru Tropical Botanic Garden and Research

Institute Herbarium (TBGT).

Extraction

The dry leaf powder of the 32 Memecylon species

(10 g each) were extracted with methanol using a

Soxhlet apparatus for 4 h. The vacuum dried extracts

were stored at 4o C until further analysis.

Estimation of total phenolic compounds

Modified Folin-Ciocalteu reagent colorimetric

method was used for the determination of total

phenolics37

. The methanol extract (0.5 mL) was

mixed with 5 mL Folin- Ciocalteu reagent (1:10

dilution with distilled water). After 5 min, 4 mL of

20 % sodium carbonate solution was added. The

mixture was allowed to stand for 15 minutes and the

absorbance was measured at 765 nm. Total phenolics

were estimated as gallic acid equivalent (GAE) mg/g

dry weight of the extract from the calibration curve

using standard gallic acid (10 -50 µg) in methanol.

Estimation of total flavonoids

Aluminium chloride colorimetric method was used

for total flavonoids determination38

. Methanol extract

(0.5 mL) was mixed with 1.5 mL of methanol, 0.1 mL

of 10 % aluminum chloride, 0.1 mL of 1 M potassium

acetate, 2.8 mL of distilled water and left at room

temperature for 30 minutes. Absorbance of the

reaction mixture was measured at 415 nm. Total

flavonoids were estimated from the calibration curve

using standard quercetin (10-50 µg) in methanol and

the results are expressed as quercetin equivalent mg/g

dry weight of extract.


367

DPPH radical scavenging assay

Radical scavenging activity was determined by

spectrophotometric method based on the reduction of

the stable free radical 2, 2-diphenyl- 1-picrylhydrazyl

(DPPH)39

. Diluted methanol extracts were added at

an equal volume to a methanolic solution of DPPH

(200 µM DPPH). After 15 min. at room temperature,

the absorbance was recorded at 517 nm. The activity

was measured as percentage, calculated using the

formula:

% scavenging = [(Acontrol-Asample)/Acontrol] x 100.

The activity was reported as IC50 value; the

concentration of sample (µg/mL) required to scavenge

50 % of DPPH radical, relative to the control (DPPH

without antioxidant agents added). Ascorbic acid was

used as positive control.

Antibacterial assay

Antibacterial activities of the extracts were

screened by the disc diffusion assay40

and the

activities were reported as diameter of the inhibition

zones that includes the disc diameter (6 mm) also.

The concentrated methanol extracts were redissolved

in DMSO and 10 µL of the DMSO extracts

(10 mg/mL) were spotted on Whatman No. 1 filter

paper discs of 6 mm diam. and aseptically applied to

the surface of agar plates at well-spaced intervals.

Authentic strains of Gram-positive [Bacillus subtilis

(MTCC 741), B. cereus (MTCC 430), Staphylococcus

aureus (MTCC 96)] and Gram-negative [Klebseilla

pneumonia (MTCC 109), Pseudomonas aeruginosa

(MTCC 741), Escherichia coli (MTCC 443), Proteus

vulgaris (MTCC 426), Serratia marcescens (MTCC

97), Salmonella typhii (MTCC 733)] bacteria obtained

from Microbial Type Culture Collection (MTCC),

maintained at the Institute of Microbial Technology,

Chandigarh, India were used for the assays. Control

discs impregnated with 10 µL of the solvent DMSO

and the standard drug streptomycin (2 µg per disc)

were used alongside the test discs in each experiment.

Statistical analysis

The experiments were replicated thrice and the

results are expressed as mean ±SD.

Results and Discussion

Phenolic and flavonoid contents

The total phenolic content expressed as mg

GAE/g of the Memecylon species are presented in

Table 1. The method provides a simple test for

determination of total phenolic content of plants due

to its high sensitivity and reproducibility. All the

Memecylon species studied contained phenolics

and are distributed in differential quantities. The

total phenolic content varied from 05.04 mg/g

(M. depressum Benth.) to 89.86 mg/g (M. gracile

Bedd.). Total phenolic content was greater than

50 mg/g extract for M. gracile (89.86 mg/g),

M. sessile Benth. (82.83 mg/g), M. deccanense

Clarke (67.06 mg/g), M. terminale Dalzell

(65.96 mg/g), M. parvifolium Thw. (64.10 mg/g),

M. heyneanum Benth. (61.06 mg/g), M. lawsonii

Gamble (59.96 mg/g) and M. mundanthuraianum

Viswanathan & Manik (51.76 mg/g).

Total flavonoid content was measured spectrophotometrically based on aluminium chloride

complex formation. The flavonoid concentration in the methanol extracts of the dried leaves of Memecylon species in terms of mg quercetin equivalent (QE) per g leaf methanol extract are presented in Table 1. The highest content of flavonoids was found in M. grande Retz. (39.56),

followed by M. capitellatum L. (31.93) and

M. bremeri (20.33). The lowest flavonoid content was found in M. talboltianum Brandis (07.60 mg/g).

Antioxidant activity of the extracts

The DPPH method is a simple, rapid and sensitive

method accepted widely for screening of samples

for radical scavenging activity41

. Table 1 shows

the quantity of each extract needed for 50%

scavenging of the DPPH free radical (IC50). IC50

of the standard compound quercetin was found to

be 10.16 (±1.75) µg/mL. The highest radical

scavenging activity was shown by M. heyneanum

Benth. (IC50 15.30 µg/mL), followed by M. sessile

Benth. (IC50 40.10 µg/mL) and M. sisparense Gamble

(IC50 44.06 µg/mL), while the activity was least for

M. terminale Dalzell (IC50 232.47 µg/mL).

Of the different class of secondary metabolites,

phenolic compounds and particularly flavonoids are

important considering their effects on human nutrition

and health are considerable. The beneficial effects

of phenolic compounds have been attributed mainly

to their antioxidant activity42

. Free radicals are

involved in many disorders like neurodegenerative

diseases, cancer and AIDS. Antioxidants through their

scavenging activities are useful for the management

of those diseases. Currently available synthetic

antioxidants are causing serious side effects and

secondary metabolites from plants are emerging as


368

promising substitutes for the synthetic ones. The

search for natural antioxidants of dietary, cosmetic

and pharmaceutical potential has become a major

industrial and scientific pursuit recently. Antibacterial activity

The antibacterial activity of different species of Memecylon extracts were tested against a group of clinically important bacterial strains by disc diffusion

assay and the results are reported in Table 2 as zone of inhibition (mm). The results revealed

moderate to good activity against the tested bacterial strains, especially for M. deccanense, M. terminale,

M. heyneanum, M. clarkeanum and M. sessile. The highest inhibition zone (26 mm) was shown by M. agastyamalaianum Santhosh et al against Bacillus

subtilis and Proteus vulgaris, M. heyneanum and M. deccanense Clarke against Proteus vulgaris

and M. parvifolium against Escherichia coli.

M. deccanense showed remarkable activity against all the tested organisms, with greater zone of inhibition than the standard drug at the tested concentrations,

Table 2—Antibacterial activity of methanol extracts of leaves of Memecylon species [Zone of inhibition (mm) by disc diffusion assay]

M. sp ID* B. c S. a B. s E. c K. p P. a P. v S. t S. m

M 1 9 9 26 9 18 13 26 21 12

M 2 12 9 24 11 18 9 14 9 9

M 3 11 8 12 8 16 9 13 11 10

M 4 12 10 14 9 16 14 11 8 8

M 5 10 8 14 10 10 9 11 9 NA

M 6 16 9 21 14 24 17 18 18 11

M 7 20 11 11 9 9 10 10 9 9

M 8 22 15 21 13 16 11 20 19 10

M 9 19 18 18 14 16 14 26 18 20

M 10 10 9 11 8 9 11 9 9 11

M 11 10 10 10 8 14 12 9 9 9

M 12 9 9 9 8 10 12 10 9 17

M 13 18 18 16 18 18 20 18 11 10

M 14 10 9 12 10 14 14 22 10 9

M 15 10 11 21 10 12 11 14 8 8

M 16 9 9 14 NA 16 11 22 9 10

M 17 10 10 11 21 14 13 11 9 10

M 18 21 20 20 19 24 21 20 17 18

M 19 11 9 14 9 16 9 9 10 9

M 20 10 9 12 10 10 11 10 9 8

M 21 10 10 10 9 11 12 13 9 11

M 22 9 9 12 9 11 12 16 9 23

M 23 12 9 13 9 12 13 12 9 9

M 24 9 11 16 16 11 16 20 8 13

M 25 10 9 11 9 14 11 14 9 10

M 26 9 8 13 10 9 10 11 9 6

M 27 21 22 24 24 24 24 26 24 23

M 28 11 9 10 11 12 9 11 8 11

M 29 9 9 20 26 18 21 20 12 12

M 30 16 10 24 10 9 11 18 8 11

M 31 11 14 16 12 10 13 14 8 11

M 32 10 11 11 NA 10 11 NA 11 7

Std 17 22 21 10 16 15 20 16 17

* Leaves methanol extracts of Memecylon species 100 µg/disc (see Table 1 for species name). B. s: Bacillus subtilis; B. c: Bacillus

cereus and S. a: Staphylococcus aureus (Gram-positive bacteria) and K. p: Klebseilla pneumonia, P. a: Pseudomonas aeruginosa,

E. c: Escherichia coli, P. v: Proteus vulgaris, S. m: Serratia marcescens, S. t: Salmonella typhii (Gram-negative bacteria).

Zone of inhibition in mm; Std: Standard antibacterial compound streptomycin (2µg/disc). NA: No activity.


369

while M. terminale showed greater zone of inhibition than the standard drug against all except Staphylococcus aureus, Bacillus subtilis and Proteus

vulgaris. M. sylvaticum Thw. and M. flavascence

Gamble showed no inhibition against Escherichia coli,

Proteus vulgaris and Salmonella marascens. All other samples showed inhibition in the range 7-25 mm against all the tested organisms. The present investigation revealed remarkable antibacterial activities of the Memecylon species against the tested bacterial strains. The screening of plants for

antimicrobial activity assumes particular significance in the context of recent emergence of infectious diseases and an increased resistance of some pathogenic microorganisms to the existing drugs.

Among the Memecylon species studied, the most

common are M. umbellatum Burm., M. malabaricum

(syn. M. randerianum) and M. edule Roxb. Though

the total flavonoid content as well as phenolic content

was less for M. umbellatum, the methanolic extract

of the leaves showed remarkable free radical

scavenging activities with IC50 value of 62.43 µg/mL.

The methanol extract of M. umbellatum showed

broad spectrum antibacterial activity with remarkable

activity against Bacillus subtilis, Klebseilla pneumonia,

Pseudomonas aeruginosa, Proteus vulgaris and

Salmonella typhii. Recent report on the antimicrobial

activity of the petroleum ether, chloroform and

ethanolic leaf extracts of M. umbellatum also showed

concentration dependent activity against all the tested

bacteria22

. The free radical scavenging activities of

M. malabaricum was negligible (IC50 207. 24 µg/mL),

whereas the antibacterial activity was moderate

with high activity against Klebseilla pneumonia and

Pseudomonas aeruginosa. It has been reported that

the methanol extract of the leaves of M. malabaricum

has activity against Gram positive, Gram negative and

fungal strains30, 31

. For M. edule, the antioxidant

activity was good with IC50 value 82.43 µg/mL and

the antibacterial activity was moderate, with highest

activities against Klebseilla pneumonia, Pseudomonas

aeruginosa and Salmonella typhii. Among the

four rare and endangered species, M. sisparense

showed good antioxidant activity with IC50 value

of 44.06 µg/mL, while the antibacterial activity was

remarkable for M. lawsonii.

Conclusion The preliminary phytochemical and pharmacological

evaluation of the hitherto uninvestigated Memecylon

species of the Western Ghats that includes rare,

endangered and endemic species, showed remarkable

antibacterial activity and presence of phenolic and

flavonoid compounds and moderate to good free

radical scavenging activities, suggesting elaborate

investigation of the genus for isolation and

characterisation of constituents and evaluation of the

biological activities. Present study also highlights the

potential of the extensive floristic wealth of the

Western Ghats region yet to be exploited.

Acknowledgements

The authors thank the Director, JNTBGRI for

providing facilities, Dr. E. S. Santhosh Kumar,

JNTBGRI for identification of the plant materials.

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