INTRODUCTION:

After four to five decades of over dependence on modern system of treatment and allopathic medication we are moving back to the elementary mode that is Ayurveda, Siddha and Unanni medicine. This back transition is evident due to inherent adverse affect of Modern system of medicine. On contrary elementary mode provides pure medication with negligible inclination towards adverse affect.

Citrus maxima (Linn) of Rutaceae family is an evergreen aromatic shrub and small trees occupies an important place in the medicine^1,2. It is also known as Pomelo. Plants are studied and proved for its analgesic and anti inflammatory activity^3,4, anti diabetic activity^5,6, CNS activity^7,8, Anti tumour activity⁹, Hepatoprotective activity^10,11, Anti bacterial activity¹², Hypocholesterolemic and ACE inhibitory activity¹³.

Hence this study brings out the pharmacognostical relevance on the plant. Which would help in the identity of the species and to maintain its standards before it intended for production or consumption. The objective of investigations was to ease the identification of the species both in whole and powdered form. The presence of valuable phytoconstituents such as flavonoids, glycosides and steroidal compounds also demand further phytochemical studies of the species.

MATERIALS AND METHODS:

Sample Collection, Processing and Storage:

Plant was collected from Cuddalore, Tamil Nadu, And authenticated for its identity by Scientist F and Head of office Dr. G.V.S. Muruthy Botanical Survey of India, Coimbatore with survey no: BSI/SR/5/23/2016/Tech/33. It was identified as Citrus maxima (Burm.) Merr. (= Aurantium maximum Burm.)-Rutaceae. The Peels of Citrus maxima were shade dried, cut into pieces, powdered and was stored in the air tight container and a portion of it was used for the Pharmacognostical and Phytochemical studies.

Macro Morphology:

Fresh peels of Citrus maxima were collected and different organoleptic features Viz shape, size, color and odour were observed. These parameters are considered useful in the qualitative control of the crude drug and evaluated as per standard WHO guideline.

Figure 1: Whole Plant

Figure 2: Leaves

Figure 3: Various parts of C.maxima

Figure 4: Seeds

Figure 5: Pulp

Figure 6: Fresh peel

Figure 7: Dried peel

MICROSCOPY:^14-16

Peels of the Citrus maxima were cut and fixed in the FAA solution (Formalin, Acetic acid and Ethyl alcohol). After fixation it is dehydrated with Tertiary butyl alcohol. The specimen is embedded in the paraffin and sectioned with the microtome, dew axed and stained with the suitable stains like toluidine blue, safranin, Fast green and iodine.

POWDER MICROSCOPY:

Shade dried peels of Citrus maxima fruits were powdered well and passed through sieve no 60 and used for the analysis. Organoleptic characters were studied using various staining reagents Viz 1% Phloroglucinol in 90% ethanol, Conc. HCl and N/50 Iodine. Slides were observed under the microscope.

FLORESCENCE ANALYSIS^17-18:

Dried powdered peel were treated with various chemical reagents and various extracts of the Citrus maxima peels were exposed to visible, ultraviolet light to study their florescence behaviour.

PHYSICOCHEMICAL PARAMETER^19-22:

Physicochemical values such as ash value, extractive value, Loss on drying, and Foaming index were determined according to the well established protocols.

PHYTOCHEMICAL SCREENING^23-25:

Aliquot of the hexane, ethyl acetate, ethanol and aqueous extracts were subjected to various phytochemical analysis using standard test procedures and reported.

Estimation of the Total Flavonoid Content^26-28:

Total flavonoid content of various extracts were determined using aluminium chloride colorimetric method for all the extracts taking Quercetine as standard

RESULTS AND DISCUSSION

MACROSCOPY:

S.no	Parameters	Observation in fresh peel	Observation in Dried peel
1	Colour Outer surface Inner surface	Yellowish green White	Yellowish brown Brown
2	Taste	Sour and Bitter	Sour and Bitter
3	Odour	Aromatic	Less aromatic
4	Surface	Plain	Spongy
5	Thickness	3 cm	1 cm

MICROSCOPY:

Fruit ranges from nearly round to oblate or pear-shaped; 10-30 cm wide, the peel were clinging or more or less easily removed, may be greenish-yellow or pale-yellow, minutely hairy, dotted with tiny green glands; 1.25-2 cm thick, the albedo soft, white or pink.

Transverse section of Citrus maxima peel:

Peels are leathery and thick in nature. The transverse section of the peel showen the presence of the epidermal cell, parenchymal cells, Collenchymatous cell and vascular bundle.

Epidermal cells:

Cells are present at the outer layer of the peel. They are arranged without any intracellular spaces.Fig:8

Figure 8: TS of Citrus maxima peel 10 X

EP- epidermis, VS- vessels, OG- oil globules

Figure 9: TS of Citrus maxima peel 20X

EP- epidermis, VS- vessels, OG- oil globules

Parenchymatous cell:

Arranged horizontally along with the epidermal cells and oil glands and oil globules are arranged in these parenchymatous layer. Fig: 10

Figure 10: TS of Citrus maxima peel 40X

PC- parenchymatous cells, XY- Xylem.

Vascular bundle:

Xylem and Phloem are arranged found in the peel where meta xylem are found at the periphery. Fig: 11

Figure 11: TS of C. maxima peel showing vascular bundle

XY- Xylem, PH- Phloem, CL- Collenchymatous cell

Collenchymatous cells:

These are present in the middle layer of the peels without any intracellular spaces. Fbres are present in these layers. Fig: 12

Figure 12: TS of Citrus maxima peel 100X

CL- Collenchymatous cell.

Crystal sheath and calcium oxalate are present in the parenchymatous layer of the peel.

Figure 13: TS of Citrus maxima peel 100X

CAO- calcium oxalate crystals.

Figure 14: TS of Citrus maxima peel 100X

SC- stone cells.

Figure 15: TS of Citrus maxima peel 100X

SC- Sclerenchymatous cell, OG- Oil globules.

Figure 16: TS of Citrus maxima peel 100X

FB- Fibres.

Figure 17: TS of citrus peel 100 x showing vessels and crystal sheath

CS- crystal sheath

POWDER MICROSCOPY:

Presence of the fibers, xylem vessels, stone cells and calcium oxalate, oil cells were also observed.

Figure 18: Oil Globules

Figure 19: Fibres

Figure 20: Cluster of stone cell

Figure 21: Calcium oxalate crystal

Figure 22: Aleurone grains

Figure 23: Fibres and vessels

FLUORESCENT ANALYSIS:

Fluorescence characteristics of the powdered peel of Citrus maxima.(table-1)

Table 1: Fluorescence characteristics of the powdered peel of Citrus maxima.

TREATMENT	VISIBLE	SHORT UV (254 nm)	LONG UV (365 nm)
Drug	Pale white	Dark brown	Orange
Drug + Water	Light yellow	Light yellow	Orange
Drug + 1 N Hcl	Light yellow	Dark green	Pale yellow
Drug + 1 N HNO₃	Light yellow	Dark green	Brownish yellow
Drug + 1 N H₂SO₄	Reddish brown	Blackish brown	Dark brown
Drug + Acetic acid	Light yellow	Pale green	Yellowish brown
Drug + 1N NaOH	Dark yellow	Greenish brown	Orange
Drug + 1N Alc NaOH	Dark yellow	Greenish brown	Orange
Drug + 1 N KOH	Dark yellow	Greenish brown	Pale brown
Drug + 1N Alc KOH	Dark yellow	Greenish brown	Pale brown
Drug + Ammonia	Dark yellow	Dark green	Dark orange
Drug + Iodine	Brownish orange	Brownish green	Reddish green
Drug + Fecl₃	Brownish green	Brownish green	Dark green
Drug + Picric acid	Dark yellow	Dark green	Brownish green

Table 2: Florescence characteristics of the extracts of the peel of Citrus maxima.

EXTRACT	VISIBLE LIGHT	SHORT UV (254 nm)	LONG UV (365 nm)
Hexane	Pale brown	Reddish brown	Brown
Ethyl acetate	Pale brown	Brownish red	Brown
Ethanol	Brown	Reddish brown	Brown
Aqueous	Brownish black	Blackish green	Dark Brown

The powdered peel and extracts of Citrus maxima showed the absence of the fluorescence characters.

PHYSIOCHEMICAL PARAMETERS

Table 3: Physiochemical parameters of Citrus maxima peels.

PARAMETERS	VALUES
Total ash	3.19% w/w
Water soluble ash	0.66% w/w
Acid insoluble ash	0.83% w/w
Loss on drying	5.23%
Foaming index	NIL
Volatile oil content	6% v/w

EXTRACTIVE VALUE

Table 4: Extractive value of the Citrus maxima peels

EXTRACTS	YIELDS %w/w
Alcohol soluble extractive	29.26% W/W
Water soluble extractive	10.25 % W/W

PHYTOCHEMICAL ANALYSIS:

Table 5: Phytochemical analysis of the various extracts.

SNO	TESTS	POWDER	HEXANE	ETHYL ACETATE	ETHANOL	AQUEOUS
1	Alkaloids	-	-	-	-	-
2	Flavonoids	+	+	+	++	+
3	Phytosterols	+	-	-	+	-
4	Triterpenoid	+	-	+	+	+
5	Tannins and phenolic compound	+	-	+	+	+
6	Saponins	+	-	-	+	+
7	Gums and mucilage	+	-	-	+	+
8	Carbohydrates	+	-	-	+	+
9	Glycosides	-	-	-	-	-
10	Proteins & amino acids	-	-	-	-	-
11	Steroids	+	-	+	+	+
12	Fixed oils and Fats	-	-	-	-	-

+ Indicates presence, - indicates Not determined

TOTLA FLAVANOID CONTENT

Table 6: Determination of Total Flavanoid content

S.NO	CONCENTRATION OF STANDARD SOLUTION(µG/ML)	ABSORBANCE(765NM)
1.	2	0.07
2.	4	0.10
3.	6	0.13
4.	8	0.16
5.	10	0.20
6.	Ethyl Acetate	0.12
7.	Ethanol	0.17

CONCLUSION:

In the present study the process of standardisation is achieved by stepwise pharmacognostical works. Which helps to measure the purity, quality and sample identification of the medicinal plants. The pharmacognostic constanst, diagnostic microscopic features and the numerical standards are reported in this work could be useful for the preparation of monograph of this plant.

REFERENCE:

1. Wealth of India, a Dictionary of Indian Raw Materials and Industrial Products Vol -3 Ca-Ci, 613.

2. Khare CP, Indian Medicinal Plants, An Illustrated Dictionary, New Delhi: Springer (India) Private Limited; 2007(1).

3. Shivananda A, Muralidhara Rao D, and Jayaveera Kn. Analgesic and anti-inflammatory activities of Citrus maxima (J. Burm) Merr in animal models. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2013;4(2):1800-1810.

4. Kundusen et. al. Exploration of Anti-inflammatory potential of Citrus limetta Risso and Citrus maxima (J. Burm.) Merr. Pharmacologyonline 2011;1: 702-709.

5. Abdul Muneer Mt, Ashok Shenoy, Karunakar Hegde, Sayed Aamer and Ar. Shabaraya. Evaluation of the anti-diabetic activity of ethanolic extract of Citrus maxima stem bark. International Journal of Pharmaceutical and Chemical Sciences 2014;3(3): 642-650.

6. A. Oyedepot and S.O. Babarinde. Effects of shaddock (Citrus maxima) fruit juice on glucose tolerance and lipid profile in type-ii diabetic rats. Chem Sci Trans., 2013;2(1):19-24.

7. Haja Sherief Sheik, Niraimathi Vedhaiyan, Sengottuvelu Singaravel. Evaluation of central nervous system activities of Citrus maxima leaf extract on rodents. J App Pharm Sci, 2014; 4(09): 077-082.

8. Vikram H. Potdar, Swati J. Kibile, Evaluation of antidepressant-like effect of Citrus maxima leaves in animal models of depression. Iranian Journal of Basic Medical Sciences 2011;14(5)478-483.

9. Sriparna Kundusen. Antitumor activity of Citrus maxima (Burm.) Merr. leaves in ehrlich's ascites carcinoma cell-treated mice. Isrn Pharmacology 2011, Article Id: 138737.

10. Sriparna Kundusen et.al., Exploration of in vivo antioxidant potential of Citrus maxima leaves against paracetamol induced hepatotoxicity in rats. Pelagia Research Library Der Pharmacia Sinica 2011;2(3): 156-163.

11. Mohammed Riaz Hasan Chowdhury et.al., Supplementation of Citrus maxima peel powder prevented oxidative stress, fibrosis, and hepatic damage in carbon tetrachloride (CCl4) treated rats. Evidence-Based Complementary and Alternative Medicine 2015, 1-10.

12. Barrion, A.S.A., et al. Phytochemical composition, antioxidant and antibacterial properties of pummelo (Citrus maxima (Burm.)) Merr. against Escherichia coli and Salmonella typhimurium. Food and Nutrition Sciences, 2014;5:749-758.

13. Ganiyu Oboh, Fatai O. Bello, Ayokunle O. Ademosun, Hypocholesterolemic properties of grapefruit (Citrus paradisii) and Shaddock (Citrus maxima) Juices and Inhibition of Angiotensin-1-Converting Enzyme Activity. Journal of Food and Drug Analysis 2014; 22:477-484.

14. Kokate CK. Practical Pharmacognosy, 4^th ed. Delhi: MK. Jain for Vallabh Prakasan, 2002. Pg.No.123-124.

15. Evans WC. Trese and Evans Pharmacognosy. 15^th ed. Rajkamal Electric Press, 2005. Pg.No.545-547.

16. The Ayurvedic Pharmacopoeia of India, New Delhi: The Controller of Publications, 2001. Pg.No.143.

17. Chase CR, Pratt RJ. Fluorescence of powdered vegetable drugs with particular reference to development of a system of identification, American Pharmaceutical System Science,1949:28. pg.no.324-333.

18. Kokosi CJ, Kokoski RJ, slama FT. Fluorescence of powdered vegetable drug under ultraviolet radiation. Journal of American Pharmaceutical Association, 1958:47.Pg.No.715-717.

19. The Ayurvedic Pharmacopoeia of India. New Delhi: The Controller of Publications; 2005; Pg.No.143

20. Indian Pharmacopoeia. New Delhi: The Controller of Publications; 1996; Pg. No. A47-60

21. Mukarjee PK, Quality Control of Herbal Drugs. New Delhi; Business Horizons; 2002: Pg.No188-399.

22. British Pharmacopoeia. General Medical Council, London: Pharmaceutical Press; 1968: pg.no.1276-1283.

23. Tyler VE, Brady LR, Robbers JE. Pharmacognosy. 9^th edition, Philadelphia USA: Lea and Febrigoree, 1998. Pg.No.78.

24. Khandelwal KR., Practical Pharmacognosy, Pune: Nirali Prakashan, 1998.Pg.No.20-42.

25. Wallis TE. Textbook of Pharmacognosy. CBS Publishers and Distributers; New Delhi, 1985.Pg.No.133-248.

26. Edeiga HO, Okwu DE, Mbaebie BO. Phytochemical constituents of some Nigerian medicinal plants. African Journal of Biotechnology, 2005:4(7).Pg.No.685-688.

27. Singleton VL, Orthofer R, Lamuela-Raventos RM. Analysis of total phenols and other oxidation substrates and Anti-oxidants by means of Folin-Ciocalteu reagent Methods Enzymol, 1999. Pg.No. 152-178.

28. Boham AB, Kocipai AC. Flavonoid and condensed tannins from leaves of Hawaian Vaccininum vaticulum and viacalycinium. Pacific Science, 2007:48. Pg.No.458-163.

Received on 30.12.2015 Modified on 22.01.2016

Res. J. Pharmacognosy & Phytochem. 8(1): Jan.- Mar. 2016; Page 25-31

DOI: 10.5958/0975-4385.2016.00006.6