INTRODUCTION:

Recent widespread interest in plant-derived drugs reflects its recognition of the validity of many traditional claims regarding the values of natural products in health care. For quality control of traditional medicines, phytochemical investigations are mainly applied. Thus, it makes a great significance to investigate chemical constituents and study pharmacological activity on this plant for its medicinal uses, which will be very useful in the field of medicine as new emerging drug. According to the WHO, medicinal plants are the best sources to obtain a variety of new herbal drug^[1].

Excoecaria agallocha Linn is a small mangrove tree that grows widely in the tidal region forests and swamps.

This plant found in the countries of temperate and tropical Asia, Australasia and South-Western Pacific^[2]. Mangrove forests are among the world’s most productive ecosystems. They enrich coastal waters, yield commercial forest products, protect coastlines, and support coastal fisheries. However, mangroves exist under conditions of high salinity, extreme tides, strong winds, high temperatures and muddy, anaerobic soils. There may be no other group of plants with such highly developed morphological, biological, ecological and physiological adaptations to extreme conditions. Mangrove forests are still quite unfamiliar to a vast population due to their limited distribution. However, the people inhabiting areas near mangrove forests heavily depend on these forests to meet their needs including their healthcare. During the early stage of human civilization, mangrove forests drew very little or no attention. This is to some extent because of the difficulty to access these areas. As the population continued to grow, people had to find new and unexplored sources including mangrove forests.^[3]

Plants that live in mangrove ecosystem are adopted to encounter high salinity, tidal extremes and heavy winds^[4]. India with a long coastline of about 7516.6 km, including the island territories has a mangrove cover of about 6,749 km2, the fourth largest mangrove area in the world. The plant exhibited antimicrobial, antihyperglycemic, anti-inflammatory, antiulcer, antinociceptive, Gastroprotective and antibacterial activities. A decoction of the leaves is occasionally given in epilepsy also.

The marine resources are nowadays widely studied because of the oceans cover more than 70% of the world surface. The terrestrial resources have been greatly explored and thus academic and industry researchers are striving to get lead molecules from the inner space of oceans.^[5]

MATERIAL AND METHODS:

Plant material:

The leaves of Excoecaria agallocha Linn (Euphorbiaceae) (EA) was collected from estuarine water at Gorai Creek, Borivali, Mumbai. The identification and authentication of these leaves were done by Botanical Survey of India (Ministry of Environment and forest). It was confirmed that, the leaves of Excoecaria agallocha L. and belonging to the family Euphorbiaceae (Vide authentication letter No: BSI/WRC/Tech./2014: dated16/09/2014; Voucher Specimen Number- MKS-1.) These identified and authentified leaves were used for the further studies.

Macroscopic study:

External features, dimensions and organoleptic properties of plants were studied. The morphological or organoleptic characters mean to the study of external appearance of plant materials. Morphological evaluation identifies the degree of purity of such crude drugs. Morphological evaluation is based on shape, size, color, surface characteristics, texture and appearance of the cut surface which are useful for the identification of plant material. The organoleptic evaluation reveals characteristics drawn from impressions^[5].

Microscopic study and Leaf constants^[6,7,8]:

The microscopic study involved the transverse section (T.S) of the leaves, leaf constants and powder microscopy. The T.S and powder microscopy were studied by staining the sample using phloroglucinol and conc. HCl (1:1) which identify the different parts of the T.S and powder. Microphotography on different magnifications was carried out with compound and digital microscopic unit. The photographs of different magnification were taken. The leaf constants such as Stomatal number, Stomatal Index, Palisade Ratio, Vein Islet no. and vein termination number of the leaf sample was determined according to the reported methods.

Determination of physicochemical constants:

The various physicochemical values of leaf powder such as Total ash value, acid insoluble ash value, water soluble ash value, Alcohol soluble extractive values, water soluble extractive value and loss on drying were determined according to the pharmacopoeial methods^{[9, 10]}.

Extraction of crude Drug:

Extraction of Excoecaria agallocha Linn:

Leaves were dried under shade, powdered and passed through mesh sieve, weighed and then used for extraction. The weighed powder (500gm) was placed in thimble made up of cellulosic filter paper and continuously extracted with Soxhlet apparatus for 72 hours using ethanol. The resulting extract was concentrated under reduced pressure using rotary vaccum evaporator to get the syrupy viscous mass. The viscous mass was transferred in porcelain dishes and dried. The amount of extract obtained was calculated for the percentage yield of the extract.

Phytochemical Screening:

Preliminary Phytochemical Screening:

Extracts obtained was subjected for qualitative chemical tests in order to identify the class of compound present^[6]. The phytochemical screening was performed on the crude extract of (ethanolic extract) of Excoecaria agallocha. One gram of the extract was dissolved in 100 ml of ethanol and was subjected to preliminary phytochemical screenings for determining nature of phytoconstituents present. Phytochemical tests were performed for the detection of the presence of different chemical constituents such as alkaloids, glycosides, flavonoids, terpenoids, carbohydrates, proteins, tannins, and other substances which are responsible for the biological activity.

Thin-layer chromatography (TLC):

TLC is the method mainly uses to investigate the presence of chemical constituent qualitatively in the plant extract. It is used to investigate alkaloids, glycosides, triterpenoids, steroids, saponins, phenolic compounds etc. It is an easy, versatile and reliable method to establish authenticity, identity and purity. The ethanolic extract of Excoecaria agallocha was subjected to TLC studies, to find the presence of number of compounds which support by the chemical test. Thin layer chromatography of Excoecaria agallocha was performed in different solvent system like, Toluene: Ethyl Acetate: Diethylamine (70:20:10); Dioxane: Ammonia (90:10) and Toluene: Ethyl Acetate: Glacial Acetic acid: Water (100:11:11:26)^{[11, 12, 13, 14, 15]}.

RESULT AND DISCUSSION:

Pharmacognostic studies: Macroscopy:

Fig.1: Leaves of Excoecaria agallocha Linn.

Colour and Appearance:

The leaves are simple with varying in size and shape, are oblong with tips varying from rounded to acuminate. The leaves are opposite while the petiole measures 1.2cm-1.4cm long; blade elliptic, 7-18 by 2-5 cm, length/width ratio 3.4-3.6, coriaceous, symmetric, base cruneate, apex acute to shortly acuminate, nerves 14-30 till apex. After twigging the leaf from stem, the plant secrets the white milky latex which is very harmful to human eye. Taste: Bitter; Odour: Aromatic.

Microscopy:

The T.S. of leaf showed the presence of upper and lower epidermis contains single layered cells. The palisade cells are single layered, compact, radialy elongated. Thin walled loosely arranged spongy parenchyma is present. Trichomes are absent on upper and lower epidermis. Vascular bundles contain lignified xylem and phloem present at the middle portion. Multilayer thick walled collenchyma present at ventral side.

Fig. 2 T.S. of leaf of Excoecaria agallocha Linn.

Powder characteristics:

The microscopic examination of powdered leaf material was performed to detect and establish various identifying microscopic characters which will be helpful in the differentiation of the substitute of the drug supplied in the form of dried powder. The photomicrographs of the identifying features of the plant material are shown in Figure 3a & 3b. Powder microscopy showed the presence of Starch grains, reticulate venation etc. Abundant starch granules are present. Starch granules are spherical and showing hilum and striations.

Fig. 3a. Powder characteristics (Starch Grains)

Fig. 3b. Powder characteristics (reticulate venation)

Quantitative Microscopy:

Various leaf constants are used for standardisation of leaf drugs and in detection of adulterants. On the leaf surface of Excoecaria agallocha Linn. diacytic type of stomata’s were observed (Fig. No. 4). Leaf constants such as Stomatal Index, Vein Islet number, vein termination number and palisade ratio are more significant in the evaluation of leaf drugs. Observed values of leaf constants are mentioned in table number 1.

Fig. 4 Stomata (Diacytic) on the leaf surface of Excoecaria agallocha Linn.

Table No. 1. Leaf constants

Sr. No.	Leaf constant	Observations
1.	Stomatal Index	10.80 to 17.20
2.	Vein islet Number	20.00 to 27.00
3.	Vein Termination Number	07.00 to 11.00
4.	Palisade Ratio	01:08 to 01:12

Physicochemical Evaluation:

The air-dried powdered leaf material was subjected for the determination of various physicochemical standardization parameters as per the WHO guidelines. The results and particulars of physicochemical evaluation parameters are given in table no. 2.

Table. 2: Physicochemical Evaluation Parameters

Sr. No.	Particulars	Result obtained (%w/w)
1.	Moisture content	0.20
2.	Total ash value	16.00
3.	Acid insoluble ash value	02.75
4.	Water soluble ash value	13.35
5.	Alcohol soluble extractive value	07.52
6.	Water soluble extractive value	01.61

Extraction of the Crude Drug:

The leaves of Excoecaria agallocha Linn. were extracted with ethanol using Soxhlet Apparatus. The extract obtained was dried to yield a Dark Green colored powdery mass (6.13%).

Phytochemical screening:

Phytochemical Screening of ethanolic extract of Excoecaria agallocha revealed the presence of alkaloids, steroids, terpenoids, Flavanoids, tannins and phenolic compounds. The result of Phytochemical Screening is recorded in Table no. 3. The qualitative results are expressed as (+) for the presence and (-) for the absence of phytochemicals for the detection of different chemical constituents.

Table No. 3: Phytochemical screening of Excoecaria agallocha Linn.

Sr. No.	Test	Ethanolic extract of leaves of Excoecaria agallocha Linn.
1	Test for Carbohydrates	+
2	Test for Protein/amino acid	+
3	Test for Fats/ waxes	-
4	Test for Steroids	+
5	Test for Glycosides	-
6	Test for Saponins	-
7	Test for Flavonoids	+
8	Test for Alkaloids	+
9	Test for Terpenes	+
10	Test for Tannins/Phenolics	+

Where; (+) = Present; (-) = Absent

Thin layer chromatography profile:

Thin-layer chromatography is particularly valuable for the qualitative determination of small amounts of constituents. Thin-layer chromatography is effective and easy to perform, and the equipment required is inexpensive, the technique is frequently used for evaluating herbal materials and their preparations. TLC studies of ethanolic extract of Excoecaria agallocha was performed in different solvent system (Table no. 4).

a) Alkaloids:

Solvent System 1 Solvent System 2

b) Flavonoid

Under 254 nm Under 365 nm

Fig. 5: Thin Layer Chromatographic profile of Ethanolic extract of Excoecaria agallocha Linn.

SUMMARY:

Leaves were used for Pharmacognostical, Phytochemical and Pharmacological evaluations. Macroscopic study of the leaves of Excoecaria agallocha showed that colour of the leaves were found to be Green in colour and shiny surface when mature. The leaves are spirally arranged on branches. The leaves had characteristic aromatic odour with bitter taste. When plucked from twig it secrets Milky Latex. Microscopical study showed diagnostic characters are present in the leaves of Excoecaria agallocha Linn; thin walled epidermal cells, spongy parenchymatus cells, palisade cell, lignified xylem vessels. Leaf constants stomatal Index, palisade ratio, vein islet and vein termination number were studied. Powder microscopy showed prominent presence of starch grains, reticulate venation. The preliminary phytochemical screening showed presence of carbohydrates, proteins, Alkaloid, flavonoids, steroids, glycosides and tannins/phenolics. The TLC profile showed effective separation and confirmed the presence of phytoconstituents, Alkaloids and flavonoids.

CONCLUSION:

It can be concluded that from the above study results, Excoecaria agallocha Linn. can serve as a valuable resource of pharmacognostic and phytochemical information. The present study is an attempt in the direction of standardization and preliminary phytochemical screening of Excoecaria agallocha Linn. Phytochemical characterization of plant extracts revealed the presence of alkaloids, steroids, terpenoids, tannins and phenolic compounds present in this plant. Further studies are going on this plant to isolate, identify, characterize and elucidate the structure of the bioactive compounds.

ACKNOWLEDGEMENTS:

The authors would like to thank Dr. Sohan S. Chitlange, Principal, Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune-18.

REFERENCES:

1. Agrawal O. P., Jain S. K., Sharma P., International Journal of Green Pharmacy, octdec.2017 11(4), 1-5.

2. Annonymous., The Wealth of India, A Dictionary of Indian Raw Materials and Industrial Products, New Delhi, 2002, D-E, III, 234.

3. Bulani V, Biyani K, Kale R et al. Inhibitory effect of Calotropis gigantea extract on Ovalbumin-induced airway inflammation and Arachidonic acid induced inflammation in a murine model of asthma. International Journal of Current Biological and Medical Science. 2011; 1(2):19-25.

4. Agoramoorthy G, Chen F, Venkatesalu V, Kuo D, Shea P, Evaluation of Antioxidant Polyphenols from Selected Mangrove Plants of India., Asian Journal of Chemistry; 2002, 1311-1322.

5. Yadav V.K., Irchhiya R., Ghosh A.K., Phytochemical and Pharmacognostic study, International Journal of Green Pharmacy, Jan-March 2018, 12(1), 1-9.

6. Khandelwal K. R., Techniques and Experiments, Practical Pharmacognosy, Nirali Prakashan, 27^th Edition, 2016, 11.1-11.8 and 24.1-24.4.

7. Anonymous. The Wealth of India, A Dictionary of Indian raw Materials and Industrial Products. New Delhi: CSIR, Publication and Information Directorate; 2002. p. 26-31.

8. Rukhsana A. Rub, Asma Mukadam, Javed Pinjari, Ajaz Nathani and Aaisha Sagri, Pharmacognostic and Phytochemical Evaluation of Quirivelia frutescens (Apocynaceae), Asian Journal of Research in Chemistry, 2(4), 2009, 509-512.

9. Harborne JB. Phytochemical methods in: A Guide to Modern Techniques of Plant Analysis. 3rd ed. U.K.: Chapman and Hall, ICMR; 1998. p. 56-99.

10. Mohini Upadhye, Uday Deokate, Murraya koenigii, Research Journal of Pharmacognosy and Phytochemistry, 9(3)2017, 177-181.

11. Wagner H, Baldt S. Plant Drug Analysis,2004, Singapore; 2^nd edi.; Springer- Verlag Publication: p.3-6,195-196.

12. Yadav VK, Singh AK, Chaudhari VK, Verma PK, Singh AK, Rajpoot S. TLC Studies and qualitative phytochemical investigations of seeds of Celastrus paniculata willd. Asian J Pharm Technol Innov 2014; 2:7-14.

13. T. Sundarrajan, V. Velmurugan, R.Srimathi, Phytochemical Evaluation and In Vitro Antidiabetic Activity of Ethanolic extract of Alternanthera ficodiaLinn., Research Journal of Pharmacy and Technology , 10(9), 2017, 2981-2983.

14. V.R. Ravikkumar, V. Gopal, N. Ravichandaran, P. Brindhaand T. Sudha, Phytochemical Evaluation of Zanthoxylum tetraspermum, Research Journal of Pharmacy and Technology,6(11), 2013, 1200-1206.

15. Kirteebala P. Pawar, Milind J. Bhitre, Priyanka V. Kalamkar, Mohan K. Kale, Pharmacognostical Studies on Leaves of Allamanda cathartica, Research Journal of Pharmacognosy and Phytochemistry, 7(2), 2015, 69-72.

16. Agarwal A, Gupta V, Gupta Amita and Jayalakshmi S, Phytochemical evaluation, Research Journal of Pharmacology and Pharmacodynamics, 1(3), 2009, 134-139.

17. Tauseef Shaikh, Atar Mujum, Khan Wasimuzzama, Preliminary Phytochemical Screening, Asian Journal of Research In Chemistry, 3(4), 2010, 977980.

18. Praveena. A, Sanjayan K. P., Pharmacognostic Studies of Morinda tinctoria, Research Journal of Pharmacognosy and Phytochemsitry, 10(3, 2018, 211-215.

19. Syed Shafeeq. R, Shekshavali. T, Syed Sulthan Ahamed. S. N., Review on Cassia auriculata, Research Journal of Pharmacology and Pharmacodynamics, 2018; 10(3): 141-145.

20. Skand Kumar Mishra, Habitat Economy and Society of the Tribal of Vindhyan region of India, J. Pharmacology and Pharmacodynamics.2018; 10(3): 98-102.

21. Kathiresan K., Bhingam B L., Biology of Mangroves and Mangrove Ecosystem., advances in Marine Biology., 40,81-251.

22. Mukherjee PK. Quality Control of Herbal Drugs: An approach to evaluation of Botanicals.New Delhi, Bussiness Horizons, 2008: p. 184-190.

23. Anonymous, Quality control methods for medicinal plant materials, World Health Organization, Geneva, 1998, 28-30.

24. Nadkarni, A.K., Excoecaria agallocha Linn., Indian Materia Medica, Popular Prakashan, 2009, 532-533.

25. Iyengar M. G., Nayak S.G.K., Anatomy of crude drugs, 11^th edition, 2008, 42-54.

Received on 17.10.2018 Modified on 19.12.2018

Research J. Pharm. and Tech. 2019; 12(3): 1289-1293.

DOI: 10.5958/0974-360X.2019.00216.6

Sr. No.	Solvent System	Proportion	R_f Value	Observation	Inference
1	Toluene: Ethyl acetate: Diethylamine	70: 20 :10	0.88	Orange brown spots.	Alkaloid Present
2	Dioxane: Ammonia	90: 10	0.58	Orange brown and Quenching zone. Dragendroff reagent UV at 254 nm	Alkaloid Present
3.	Toluene: Ethyl Acetate: Glacial Acetic Acid: Water	100:11:11:26	0.82	Quenching Under Long UV 365 nm	Flavonoid Present