PHYTOCHEMICAL PROFILING, ANTIBACTERIAL SCREENING AND ANTIOXIDANT PROPERTIES OF THE SACRED TREE (SHOREA ROBUSTA GAERTN.) OF JHARKHAND

PHYTOCHEMICAL PROFILING, ANTIBACTERIAL SCREENING AND ANTIOXIDANT PROPERTIES OF THE SACRED TREE (SHOREA ROBUSTA GAERTN.) OF JHARKHAND

Raphael R. Marandi ¹*, S. John Britto ² and Prabhat K. Soreng ³

Department of Botany ¹, St. Xavier’s College, Mahuadanr, Nilamber-Pitamber University, Jharkhand -822119, India

Rapinat Herbarium and Centre for Molecular Systematics ²,St. Joseph’s College (Autonomous), Bharathidasan University Tiruchirappalli, Tamil Nadu - 620002, India

Department of Botany ³, St. Xavier’s College, Ranchi University, Jharkhand - 834001, India

ABSTRACT: Shorea robusta Gaertn., commonly called as Sal tree, is revered as a sacred tree by most of the tribals of Jharkhand, India. They do not worship it rather believe it to the abode of tribal goddess. The plant parts are used as a remedy against various ailments in combination with other ingredients. Preliminary phytochemical screening of the whole plant exhibited the presence of high concentration of bioactive components. The plant extracts also exhibited good antibacterial activities. The HPLC and GC-MS analysis showed the presence of very high concentrations of several phytochemicals such as Trimethylsilyl 3-methyl-4-[(trimethylsilyl)oxy] benzoate, D-Mannitol, Sorbital, Phytol, Hexamethylcyclotrisiloxane, β-Caryophyllene, 1,2,4-Benzenetriol, etc. which are pharmaceutically and industrially very important. Several of these phytochemicals have been determined to be anti-cancerous and antioxidant, antidiarrheal, anti-dysenteric, antibacterial, stimulant, diuretic, styptic and anti-gonorrhoeal. The study revealed that the sacred tree of Jharkhand could be a real blessing for the world in order to harvest several phytochemicals in large quantities and to produce drugs at the low cost to heal several human ailments

Shorea robusta, Phytochemical, Antibacterial, Antioxidant, Jharkhand

INTRODUCTION: Sal is considered to be a sacred tree by most of the tribals of Jharkhand, India. They do not worship it but they believe it to the abode of tribal goddess Chalapachcho or Jaher era or Jaherburhi. Every tribal village possess a sacred grove (collection of Sal trees) at one end of the village in which the village deities dwell under the chieftainship of Chālāpachchoor Jaher era.

Scientifically, a Sal tree is called as Shorea robusta Gaertn. Which belongs to the family Dipterocarpaceae. It is called by different names by various ethnic groups as follows –Makka (Oraons), Sakhua (Sadri), Serga (Kharias) and Sarjom (Mundas, Santals and Hos). The sacred grove is called as Chālā (Oraons), Sarnatharo(Kharias), Jayar (Mundas), Jaher (Santals) and Sarna (Hos).

The entire tree of S. robusta is used for varied purposes such as timber in house construction, agriculture tools, firewood, twig as toothbrush, leaves for making leaf-plates and cups, flowers are offered to deities in Sarhulor Baha festival and the seeds are used as a food supplement with the boiled flowers of Mandhu calatifolia. According to the informants, the bark decoction or seed powder or gum-resin with jaggery is given to treat diarrhoea and dysentery. The bark decoction with black salt is given to manage diabetes and also to women against burning sensation during urination. The tender leaves are chewed against indigestion.

Literature review reveals that several works have done on the oleo resin of S. robusta regarding its anti-diarrhoeal, anti-dysentery, anti-skin allergic, emulsifying and antibacterial properties ^{1, 2}. It acts as stimulant, expectorant, diuretic, styptic and also has been used against gonorrhoea, bleeding piles, bronchitis, and leucorrhoea, menorrhagia, enlargement of the spleen ³. All the parts of the tree has been studied by different workers taking a single part – bark ^4-6, leaves ^7-9, flower ¹⁰ and seeds¹¹.  No cumulative works of all the parts have been reported, hence the need of the present study. Moreover, despite being the sacred and state tree of Jharkhand, no extensive works have been done on S. robusta of the state with respect to phytochemical and antibacterial studies.

Taxonomy:               

The taxonomy hierarchy of S. robusta is given in Table 1 with its common and vernacular names. S. robusta is a large, deciduous tree up to 50 m tall bearing epicormic branches and spreading or spherical crowns (Fig. 1a). The trunk girth is up to 5m consisting of thick and dark brown bark with longitudinal fissures. The trunk becomes shallow in aged trees. Leaves are simple, shiny, glabrous, about 10-25 cm long and broadly oval at the base, with the apex tapering into a long point (Fig. 1b).

The new leaves are reddish which turn delicate green and finally dark green. Flowers are yellowish-white which are arranged in large terminal or axillary racemose panicles.  Fruits are capsules, ovoid, and about 1.3-1.5 cm long and 1 cm in diameter. They are winged with enlarged sepals of 5 unequal sizes and lengths (Fig. 2c). Seeds and greenish and fleshy with unequal cotyledons.

TABLE 1: TAXONOMY OF SHOREA ROBUSTA

FIG.1: A) SHOREA ROBUSTA GAERTN. (HABIT- TREE IN FULL BLOOM B) CLOSE UP LEAVES AND FLOWERS OF S. ROBUSTA

FIG.2: A) SHOREA ROBUSTA BARK OUTER SURFACE. B) SHOREA ROBUSTA BARK INNER SURFACE C) FRUITS AND SEEDS OF S. ROBUSTA

MATERIALS AND METHOD:

Collection of Plant Materials:

The voucher specimens and the plant materials such as bark, leaves, flowers and seeds of S.robusta were collected from the jungles of Balumath, Jharkhand, India. The herbarium specimens were authenticated by Dr. S. John Brittoand were deposited in the Rapinat Herbarium of St. Joseph’s College, Trichy, Tamilnadu, India with the accession number RHT 67036 and RHT 67059. The different habits, flowers and seeds of the tree were photographed and deposited in the same herbarium. The plant parts for the experiments were collected during the months of April-May, 2015.

Extraction of Phytochemicals:

The plant parts such as bark, leaves, flowers and seeds of S.robusta were dried under shade at room temperature for a period of two weeks. The dried plant materials were powdered mechanically and kept in the air-tight containers.  10g of the powder of each plant part was extracted in a rotary shaker for 72 hours with 90% ethanol and distilled water. The extracts were concentrated and dried by evaporation.

Preliminary Phytochemical Investigations: ^12-19

A pinch of powders of bark, leaf, flowers and seeds of S.robusta taken on a test tube and added with a few drops of chemical reagents such as strong acids, strong bases and other reagents. The characteristic colours produced by the reactions were observed and recorded as per the standard methods.

Qualitative phytochemical analysis of ethanolic and aqueous extracts of bark, leaves, flowers and seeds of S.robusta was carried out by adopting standard methods from various sources. The bioactive compounds such as alkaloids, carbohydrates, flavonoids, glycosides, phenols, saponins, tannins, terpenoids, etc. were screened by doing three different tests for each phytochemical to ascertain the presence.

 Microscopic Study of Powders: ²⁰

A pinch of powders of bark, leaves, flowers and seeds of S.robusta was taken on a slide and added with few drops of safranin and glycerine. It was mixed well and then observed under microscope and photographed with Nikon Eclipse 80i. The photographs were edited with NIS Elements F 3.00 SP7and Adobe Photoshop CS6 softwares for labelling.

 Antibacterial Screening of Sacred Tree:

Twelve bacterial pathogens consisting of four Gram^+ve and eight Gram^-ve were selected for the antibacterial study of the sacred tree. The selected Gram ^+ve pathogens were Bacillus cereus(ATCC #4342), Bacillus subtilis (MTCC # 441), Staphylococcus aureus (MTCC # 3163) and Streptococcus pneumonia (ATCC # 7066), while the Gram^-ve pathogens included Enterobacter aerogenes (MTCC # 2990), Escherichia coli (MTCC# 199), Klebsiella pneumonia (MTCC # 3040), Proteus mirabilis(MTCC # 1429), Proteus vulgaris (MTCC # 1771), Pseudomonas aeruginosa (MTCC # 2474), Salmonella paratyphi (MTCC #734) and Vibrio cholerae (ATCC # 14104).

The ethanolic extracts of bark, leaf, flower and seed of S. robusta were tested for susceptibility against all the given pathogens adopting standard disc diffusion method in Nutrient agar medium. The concentrations of 200µg/disc was taken for the extracts and also the control (streptomycin). The experiments were done in triplicates from which the mean and standard deviations were calculated by using standard formulae.

 HPLC and GC-MS Analysis of Sacred Tree:

Ethanolic extracts of bark, leaf, flower and seed of S. robusta was subjected to analytical HPLC adopting the standard procedures and conditions ²¹: 2ml of extract was filtered through 0.2μm filter and 20μl was injected into the Shimadzu HPLC equipped with auto-sampler and diode array detector. The solvents Acetonitrile and HPLC grade water were used for gradient elution and the running time consisted of 30 minutes, while the chromatogram was obtained at 254nm. For the GC-MS analysis, the same extracts were subjected to GC-MS Shimadzu instrument by adopting standard procedure and conditions ^{22, 23}. Identification and interpretation of compounds were done by the comparison of mass spectra of the samples using the database of NIST research library. Spectra of unknown compounds were compared with the spectra of known compounds stored in the NIST library. The names, molecular formula, molecular weight and molecular structures of the compounds of the test extracts were ascertained from the databank of PubChem²⁴ and Chem Spider ²⁵. The biological activities of the compounds were obtained from various sources which have been referenced.

 Antioxidant Activity by DPPH Radical Scavenging Assay: ^{26, 27, 28}

Free radical scavenging activity of ethanolic extracts of bark, leaf, flower and seed of S. robusta was determined by DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay with slight modification.3ml of DPPH (30mg/L) solution was added to 1ml of sample solution at different concentrations (100-500µg/ml). The reaction mixture was mixed well and kept in the dark at room for 30minutes. The absorbance was measured at 517 nm by using Lambda 35 UV/VIS Spectrometer. The absorbance of the samples were compared with that of the control standard (Ascorbic acid). The IC₅₀ value of samples (concentration of sample required to inhibit 50% of the DPPH free radical) was calculated using Log dose inhibition curve.  The lower absorbance of the reaction mixture indicated higher free radical activity. The ability of the plant extracts to scavenge DPPH radical was calculated by the following formula:

DPPH scavenging effect (%) or Percent inhibition

= A₀ - A₁ / A₀ × 100.

Where A₀= Absorbance of control and A₁ = Absorbance of samples.

RESULTS AND DISCUSSIONS:

Phytochemical Screening: The powders bark, leaf, flowers and seeds of S. robusta, when treated with different chemical reagents produced specific colour reactions. On the basis of colour reactions, the inferences were drawn for the presence of the phytochemicals (Tables 2 and 3).  The powder studies indicated the presence of alkaloids, phenols, tannins, steroids, flavonoids, etc. in the bark, leaf, flower and seed of the sacred tree. However, protein was absent in all parts except the seed. Similarly, anthraquinone absent in bark and leaf, while present in flower and seed.

TABLE 2: BEHAVIOUR OF BARKAND LEAFPOWDERS OF S. ROBUSTAWITH DIFFERENT CHEMICAL REAGENTS

Abbreviations: Conc. – concentrated;, Aq.-Aqueous

TABLE 3: BEHAVIOUR OF FLOWER AND SEED POWDERS OF S. ROBUSTA WITH CHEMICAL REAGENTS

Abbreviations: Conc. – concentrated;, Aq.-Aqueous

Preliminary phytochemical screening of the ethanolic and aqueous extracts of bark, leaf, flower and seed of S. robusta were carried out. The dried extracts were dissolved in 15ml of respective solvents and were tested for the presence of bioactive compounds such as, alkaloids, carbohydrates, flavonoids, glycosides, phenols, steroids, tannins, saponins etc. The results are given in Table 4. The data in the table indicate that both the ethanolic and aqueous extracts contain most of bioactive phytochemicals. Moreover, carbohydrates, reducing sugars, phenols and tannins were found to be present in quite high concentrations. The bark was found to possess good concentration of flavonoids, cardiac glycosides, steroids and terpenoids. The starch and fixed oils were not detected in any plant part of S. robusta.

TABLE 4: PHYTOCHEMICAL SCREENING OF DIFFERENT PARTS OF S. ROBUSTA

Very high (++++), high (+++), moderate (++), low (+) and nil (–)

Fluorescent analysis of Extracts: ^{29, 30}

A small quantity of the extract was placed inside the UV viewer chamber and viewed in visible light and short ultraviolet radiations (254 nm). The ethanolic and aqueous extracts were observed under the visible light and UV light for their characteristic colours and the colour data were recorded (Table 5).

TABLE 5: FLUORESCENT ANALYSIS OF ETHANOLICAND AQUEOUS EXTRACTS OF S. ROBUSTA

 Microscopic Analysis of Powders:

The components observed in the bark powder of S. robusta are presented in Fig. 2a-j. The pharmacognostic markers were thick walled cork cells with wavy walls, starch grains, thick walled stone cells with broad lumen, sclereids, gum ducts, prismatic and druse crystals, crystal fibres and heterogeneous medullary rays filled with starch grains. On the other hand, the leaf powder of S. robusta exhibited unicellular trichomes, resin crystals, druse crystals of calcium oxalate, stomata, simple starch grains and oil globules (Fig. 3a-i). The flower powder of S. robusta consisted of glandular and unicellular trichomes, pollen grains, resin crystals, few starch grains and oil globules (Fig. 4a-i). The seed powder of S. robusta was found to contain abundance of spherical starch grains, oil globules and a few resin crystals (Fig. 5a-f). was interesting to note that all parts of the sacred tree consisted of resin crystals and oil globules. Even the calcium oxalate crystals were observed in all parts but the seed. A spherical structure with a nucleus in the centre was observed, which is a mystery to be identified in the sacred tree (Fig.3g & 4c).

FIG.2: a-j)  BARK POWDER MICROSCOPY OF S. ROBUSTA; Mr Medullary rays; Cf- Crystal fibre; Rs Resin crystal; Og- Oil globule; Cr- Calcium oxalate crystal; Sg- Starch grain; Fr- Fibre; Sc- Sclereid

FIG.3 a-i)  LEAF POWDER MICROSCOPY OF S. ROBUSTA; Tr- Trichome; Cr- Calcium oxalate crystal; St Stomata; Rs Resin crystal; Og- Oil globule; Sg- Starch grain;

FIG.4: a-i)  FLOWER POWDER MICROSCOPY OF S. ROBUSTA; Fr- Floral hairs; Pg- pollen grains; Cr- Calcium oxalate crystal; Rs Resin crystal; Og- Oil globule; Sg- Starch grain; GFr- Group of floral hairs

FIG.5: a-f)  SEED POWDER MICROSCOPY OF S. ROBUSTA; Rs Resin crystal; Og- Oil globule; Sg- Starch grain;

Antibacterial Activities:

The antibacterial activities of the ethanolic extracts of bark, leaf, flower and seed of the sacred tree were tested against 4 Gram ^+ve bacteria and 8 Gram^-ve bacteria using Streptomycin as control. The results are presented in Table 6 and Chart 1. All the plant parts exhibited activities all the tested microorganisms. However, they showed minimum inhibition zones against B. Subtilis and P. aeruginosa. It is clear from the analytical chart that the bark and seed possess higher antibacterial activities followed by leaf and flower. Moreover, leaf exhibited considerable sizes of zones against S. aureus and S. faecalis.  The higher antibacterial activities exhibited by the seed validates its ethnic usage against diarrhoea, dysentery and gastritis.

TABLE 6: ANTIBACTERIAL ACTIVITIES OF DIFFERENT PARTS OF. SHOREA ROBUSTA

Data given are Mean of triplicates ± Standard Deviation

CHART 1: COMPARATIVE STUDY OF ANTIBACTERIAL ACTIVITIES OF SHOREA ROBUSTA

HPLC Analytical Examination of Sacred Tree:

The HPLC analysis of ethanolic extract of bark, leaf, flower and seed S. robusta produced 1, 1, 4 and 2 peaks respectively (Fig.6-8). The details such as retention time and area percentage are given in

Table 7. It was interesting to note that the flower produce higher number of peaks followed by seed, leaf and bark. The results indicated that the flower and seed possess higher number bioactive compounds.

 Bark extract of S. robusta:

FIG. 6: HPLC CHROMATOGRAM OF ETHANOLIC

 Leaf extract of S. robusta:

FIG. 7: HPLC CHROMATOGRAM OF ETHANOLIC

Flower extract of S. robusta:

FIG. 8: HPLC CHROMATOGRAM OF ETHANOLIC

 Seed extract of S. robusta:

FIG. 9: HPLC CHROMATOGRAM OF ETHANOLIC

TABLE 7: HPLC DETECTION OF DIFFERENT PARTS OF SACRED TREE

GC-MS Data Analysis of Sacred Tree:

The bioactive compounds detected through GC-MS analysis of ethanolic extracts of bark, leaf, flower and seed of the sacred tree are presented in Table 8, 9, 10 and 11. The number of peaks produced and the compounds detected are: bark – 4, leaf – 7, flower -6 and seed – 4. The industrial uses and the biological activities of the phytochemicals are given in the respective tables. The bark of S. robusta possess high concentration of 1,2,4-

Benzenetriol, Ethyl(trimethyl)silane and D-Mannitol with the area percentage 35.20, 19.97 and 42.65 respectively. The leaf of the sacred tree possess good concentration of Trimethylsilyl 3-methyl-4-[(trimethylsilyl)oxy]benzoate, Propyl octan-2-yl carbonate and n-Hexadecanoic acid with area percentage 52.26, 21.23 and 10.11 respectively. The flower showed heavy concentration of β-Caryophyllene, Undecanal, 4a-Methyl-3,4,4a,5,8,8a-hexahydro-2(1H)-naphthalenone and Sorbitol with the area percentage 39.41, 13.74, 10.58 and 27.17 respectively while the seed exhibited the presence of high concentration of Hexadecyltrichloroacetate, Cyclooctane, methyl-2-Decanol and Hexamethyl cyclotrisiloxane with area percentage as 67.91, 25.37 and 100.00 respectively.

TABLE 8: GC-MS ANALYSIS OF BARK ETHANOLIC EXTRACT OF S.ROBUSTA

RT-Retention Time, M.F.-Molecular Formula, M.W.-Molecular Weight

TABLE 9: GC-MS ANALYSIS OF LEAF ETHANOLIC EXTRACT OF S.ROBUSTA

TABLE 10: GC-MS ANALYSIS OF SEED ETHANOLIC EXTRACT OF S.ROBUSTART-Retention Time, M.F.-Molecular Formula, M.W.-Molecular Weight, -no reference

However, the GC-MS analysis of same plant from Jharkhand revealed several new phytochemicals which are pharmaceutically and industrially important, eg. Trimethylsilyl 3-methyl-4-[(trimethylsilyl) oxy] benzoate, D-Mannitol, Sorbital, Phytol, Hexamethyl cyclotrisiloxane, etc. Moreover, these phytochemicals were detected in a very high concentration indicating that the plant parts could be rich sources of good harvest.Pharmaceutically important phytochemical such Ursolic acid, α-Amyrenone, α-Amyrin, β-Amyrin, Shoreaphenol, Hopeaphenol, Friedelin, β-Sitosterol, Dihydroxyisoflavone, Asiatic acid, Benthamic acid and Uvaol ^{47, 48} have already been isolated from different parts of the plant. Preliminary phytochemical analysis of plant from other parts of India revealed the presence of leucoanthocyanidin, hopeaphenol, triterpenoids and a terpene alcohol, furfural, monomethylether, dimethylether of homocatechol, alkybenzene derivatives, pentosans, lignan, tannin, amino acids, fatty acids, triterpenoids, ellagic, chebulinic, gallic, phenolic and shorbic acids ⁴⁹.

DPPH Radical Scavenging activities:

The scavenging activity on DPPH radicals was used to determine the free radical-scavenging activity of different parts of the sacred tree. The results are presented in the form a chart (Chart 2). The reduced antioxidant became pale yellowish in colour, which was used to evaluate the antioxidant activity of bark, leaf, flower and seed of S. robusta. It was found all the parts of the sacred tree consist of very high free radical scavenging activities. The concentrations of 100-500 μg/ml exhibited higher free radical scavenging activity than the control (Ascorbic acid). Moreover, the bark was found to possess higher antioxidant activity than the other parts of the sacred tree. Jeyadoss et al. (2014) ⁵⁰ and Nethaji et al. (2014) ⁵¹ also determined the high antioxidant capacity of S. robusta leaf. The whole plant free radical-scavenging activity is being reported for the first time.

CHART 2: DPPH FREE RADICAL SCAVENGING ACTIVITY OF S. ROBUSTA

CONCLUSION: It is first time report of the whole plant study with various scientific parameters. The results of phytochemical tests, antibacterial screening and antioxidant assay are supportive of the usage of the sacred tree to heal various ailments among different ethnic groups of Jharkhand. The antibacterial results are evident that the different parts of the tree has curative power against diarrhoea, dysentery, diabetes, burning sensation, indigestion, skin allergies, expectorant, diuretic, gonorrhoea, bleeding piles, bronchitis, leucorrhoea, menorrhagia and enlargement of the spleen. Preliminary phytochemical screening exhibited the presence of very high concentration of bioactive components such as alkaloids, glycosides, phenols, tannin, steroids and terpenoids which contribute to high antibacterial activities. The HPLC and GC-MS analysis determined the presence of very high concentrations of several phytochemicals such as Trimethylsilyl 3-methyl-4-[(trimethylsilyl)oxy] benzoate, D-Mannitol, Sorbital, Phytol, Hexamethylcyclotrisiloxane, β-Caryophyllene, 1,2,4-Benzenetriol, etc. which are pharmaceutically and industrially very important. Moreover, the phytochemical, Trimethylsilyl 3-methyl-4-[(trimethylsilyl) oxy] benzoate, found in a very high concentration in leaf of the S. robusta, has been determined to be anti-cancerous and a powerful antioxidant. This could open a new pathway for further research. Hence, the sacred tree of Jharkhand could be a real blessing for the world in order to harvest several phytochemicals in large quantities and to produce drugs at the low cost to heal several human ailments.

ACKNOWLEDGMENTS: Authors are grateful to Dr. S. John Britto (Director & Head) and the staff of Rapinat Herbarium and Centre for Molecular Systematics, St. Joseph’s College, Trichy, Tamilnadu, India. They cordially thank Rev. Tony Xavier and Sr. Prisca SND of Balumath Parish, Daltonganj for necessary help, and also the traditional healers of Jharkhand. The authors are grateful to UGC, New Delhi for financial support through RGNF fellowship.

 CONFLICT OF INTEREST: None

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    How to cite this article:

    Marandi RR, Britto SJ and Soreng PK: Phytochemical Profiling, Antibacterial Screening and Antioxidant Properties of the Sacred Tree (Shorea Robusta Gaertn.) of Jharkhand. Int J Pharm Sci Res 2015; 7(7): 2874-88.doi: 10.13040/IJPSR.0975-8232.7(7).2874-88.

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