Pharmacognostical Standardization of Leaves of Ravenala madagascariensis Sonn.

S. Sakthi Priyadarsini, R Vadivu* and N Jayshree

Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai, Tamilnadu, India.

ABSTRACT:

Standardization was carried out on the basis of detailed botanical evaluation of the leaves which includes morphology and microscopy as well as WHO recommended physico-chemical studies. The results of the standardization may throw a immense light on the botanical identity of the leaves Ravenala madagascariensis Sonn., which may furnish a basis of judging the authenticity of the plant and also to differentiate the drug from its adulterants and other species.

Ravenala madagascariensis Sonn., (Strelitziaceae), also known as Visirivazhai in Tamil, is a native of Madagascar, widely cultivated for its distinctive habit in tropical and subtropical regions. In addition to its ornamental nature, this tree has been widely used in traditional medicine for the treatment of diabetes and kidney stone problems. The seeds are reported to be antiseptic. The leaves of Ravenala madagascariensis Sonn., have close resemblance with Musa leaf. Hence pharmacognostical standardization will contribute to the differentiation of Ravenala madagascariensis Sonn. leaves from that of Musa paradisiaca. The anatomy of leaf showed squarish upper epidermis. The lower epidermis was stomatiferous. Cyclocytic stomata, druses, raphides, mesophyll with tannin bodies, lobed sclereids remain the characteristic features of the leaf microscopy. Physicochemical parameters were evaluated and fluorescence analysis were carried out.

Thus the study employs the correct identification of standardization of the drug in crude form.

KEYWORDS: Ravenala madagascariensis Sonn., Standardization, Botanical, Physico-chemical constants

INTRODUCTION:

Nowadays many ornamental plants are being upgraded by validating the Pharmacognostical standards, traditional claims and establishing its medicinal value. Various plants are yet to be scientifically proven for their identity and therapeutic efficacy. A survey of literature and screening for scientific data of many plants revealed that there is a lacking in systematic Pharmacognostical standards. So the present study is undertaken to standardize the drug Pharmacognostically which will help in the identification of the drug in crude form.

Ravenala madagascariensis Sonn., is one such fascinating ornamental plant belonging to bird of paradise family, Strelitziaceae. This elegant, striking tree is commonly known as Traveler’s palm or Traveler’s tree. (Fig. 1) It was claimed to be widely useful for the treatment of diabetics and kidney stone problems ¹Also the seeds were reported to be antiseptic². The leaves show close resemblance with that of banana (Musa paradisica) and also no reports were found upon the

pharmacognostical profile which provoked the need of its pharmacognostical standardization to study its morphology, microscopy and physico-chemical constants. The study parameters includes organoleptic features, macroscopy, microscopy, fluorescence analysis, ash values, extractive values, crude fibre content, loss on drying, foaming index, inorganic metal analysis and preliminary phytochemical analysis in order to establish its identity and purity.

EXPERIMENTAL:

Plant material:

The leaves of Ravenala madagascariensis Sonn., (Strelitziaceae) were collected from Subramaniapuram Park, Trichy District, TamilNadu, during June 2009..The plant was identified and authenticated by Botanical survey of India, Coimbatore, (No. BSI / SC /5/ 23 / 09-10 / Tech-622) and a voucher specimen has been deposited in the Department for future reference.

Macroscopic and Microscopic analysis:

The macroscopy and microscopy of the leaf was studied according to the method of Brain and Turner³. For the microscopical studies the cross sections were prepared and stained as per the procedure of Johansen⁴ and the quantitative microscopy was studied as per the procedure given by Wallis⁵ and P.K.Lala⁶. The powder analysis has been carried out according to the method of Brain and Turner⁷ and C.K. Kokate⁸.

Physico-chemical analysis:

The ash values, extractive values, crude fibre content, loss on drying and foaming index were performed according to the official methods prescribed in Indian Pharmacopoeia⁹ and the WHO Guidelines on quality control methods for medicinal plant materials¹⁰. Fluorescence analysis was carried out according to the method of Chase and Pratt¹¹ and Kokoski etal^12.

Inorganic metal analysis:

The inorganic metal analysis for the powdered leaves has been carried out by X-ray fluorescence method.¹³

Preliminary phytochemical screening:

Preliminary phytochemical screening was carried out by using standard procedures described by Kokate¹⁴ and Horborne¹⁵.

RESULTS AND DISCUSSION:

Pharmacognostical studies play a key factor in establishing the authenticity of the plant material. The botanical identity of the leaf was established by examining its anatomical features.

Taxonomy:

Ravenala madagascariensis Sonn. is the sole member of its genus and is closely related to the South African genus Strelitzia and South American genus Phenakospermum. Some older classification includes this genera in the banana family, Musaceae.

Macroscopical features:

The leaves are simple, oblong with smooth surface, 1.5 - 3 x 0.6 - 1m, retuse apex, asymmetric base and entire margin with pinnately parallel venation. (Fig. 1)

Microscopical features:

The anatomical examination of Ravenala madagascariensis Sonn., leaves exhibited important microscopical features like druses, cyclocytic stomata, calcium oxalate raphides, lobed sclereids and tannin bodies.

The lamina is smooth and are measuring 450μm near the midrib and 340μm near the margin. The T.S of lamina near the midrib is slightly different from that of the lamina along the margin.

Fig. 2 T.S OF LAMINA (Middle portion)

AdE – Adaxial Epidermis; AbE – Abaxial Epidermis; BS – Bundle sheath; HC – Hyaline cell; Ph – Phloem; PM - Palisade mesophyll; SC – Sclerenchyma; SBS – Sclerenchymatous bundle sheath; SM – Spongy mesophyll; VB – Vascular bundle; X – Xylem; SE – Subepidermal cell

Anatomy of leaf showed squarish upper epidermis with prominent cuticle and the lower epidermis was stomatiferous and thick walled. (Fig. 2). Thin adaxial epidermal layer of small squarish cells was present with a prominent cuticle. (Fig. 3) The epidermal layer is 10μm thick. Beneath epidermis, a zone of three or four layers of vertically stretched rectangular six sided, thin walled hyaline cells. The lower epidermis is less prominent and narrow; the cells squarish, stomatiferous and thick walled. Inner to the hyaline tissue is the palisade tissue comprising of two layers of columnar chlorophyllous cells. Spongy mesophyll tissue occurs in the median portion of the lamina. Vascular system consists of a horizontal row of vertically oblong and straight vascular bundles. The vascular bundle in middle part of the lamina is the largest and those on either side are smaller.

The midrib is semicircular with adaxially folded wings of the lamina. Three systems of vascular bundles, A row of circular, collateral abaxial vascular bundles, a median row of vascular bundles and an adaxial (inner) band of larger, vertically oblong bundles is found to be more characteristic. (Fig. 3)

Small calcium oxalate druses are seen in some of the cells of the adaxial epidermis. (Fig. 4.1) The main parallel veins are interlinked by horizontal cross-veins consists mostly of tracheids. Also the transdermal clearing showed some of the mesophyll cells containing dense masses of tannins. (Fig. 4.2)

Cr – Crystal; MV – Middle vein; PM - Palisade mesophyll; Ve - Vein

BS – Bundle sheath; AdVB – Adaxial Vascular Bundle; Ep – Epidermis; MVB – Median Vascular Bundle; Ph – Phloem; OVB – Outer Vascular Bundle; SCc – Sclerenchyma cap; SEp – Sub – Epidermis; X – Xylem.

PM – Palisade mesophyll like tissue; Scl – Sclereid

Cyclocytic stomata with one or two whorls of subsidiary cells, with each whorl containing five subsidiary cells are a characteristic feature of its botanical identity. (Fig. 5)

Powder Microscopy:

Sclereids are long, narrow and cylindrical; they are either straight or lobed are frequently seen in powder. The narrow fibres are 800μm long and 20μm wide; the wide fibres are 900μm long and 40μm wide. Calcium oxalate raphides are fairly common in the midrib.

Quantitative microscopy:

Leaf constants have a great diagnostic significance in the evaluation of leafy drugs. Quantitative microscopic data such as measurement of fibre length and width has been highly relied upon by pioneer pharmacognosist. Indeed these features can be employed for interspecific identity of drugs. The stomatal number, vein islet number, palisade ratio per sq. mm, the length and width of the fibres were given in Table. 1

TABLE -1: Leaf constants of leaves of Ravenala madagascariensis Sonn., (Strelitziaceae)

S. No	Parameters	Values in 1 sq mm
1	Stomatal number	100
2	Stomatal index	1.75
3	Vein islets number	15.3
4	Palisade ratio	7.4
5	Fibres Length\Width	800-900µm\20-40 µm

TABLE – 2: Physico-Chemical Constants of the leaves of Ravenala madagascariensis Sonn., (Strelitziaceae)

S.No	Parameters	Percentage (%w/w)
I	ASH VALUES
1.	Total ash	12.38
2.	Water soluble ash	9.78
3.	Water insoluble ash	1.799
4.	Acid soluble ash	2.783
5.	Acid insoluble ash	10.21
6.	Sulphated ash	14.103
II	EXTRACTIVE VALUES
1.	Ethanol soluble extractive	8
2.	Water soluble extractive	9.33
3.	Ether soluble volatile	2
4.	Ether soluble nonvolatile	2.93
III	LOSS ON DRYING	6.56
IV	VOLATILE OIL CONTENT	nil
V	CRUDE FIBRE CONTENT	51.926
VI	FOAMING INDEX	Less than 100
VII	SWELLING INDEX	7.6 ml/gm

TABLE – 3: Fluorescence analysis of leaf powder of Ravenala madagascariensis Sonn.,

S.No	Treatment	Day light	Short – UV (254 nm)	Long – UV (366 nm)
1	Powder	Yellowish green	Pale yellow	Dark green
2	Powder + water	Yellowish brown	Pale yellow	Dark green
3	Powder + 1N HCl	Pale yellow	Pale yellow	Pale green
4	Powder + 1N H₂SO₄	Pale brown	Pale green	Green fluorescence
5	Powder + 1N HNO₃	Pale brown	Green	Green
6	Powder + Acetic acid	Pale brown	Pale brown	Light Green fluorescence
7	Powder + 1N NaOH	Yellowish brown	Green	Green fluorescence
8	Powder+ 1N Alc. NaOH	Yellow	Green	Green fluorescence
9	Powder + 1N KOH	Pale brown	Green	Green fluorescence
10	Powder + 1N Alc. KOH	Yellow brown	Green	Green fluorescence
11	Powder + Ammonia	Brown	Pale Green fluorescence	Light Green fluorescence
12	Powder + Iodine	Pale brown	Green	Green
13	Powder + FeCl₃	Green	Brown	Green fluorescence
14	Powder + Ethanol	Yellow	Reddish Brown	Green fluorescence

TABLE – 4: Fluorescence analysis of extracts of the leaves of Ravenala madagascariensis Sonn.,

S.No	Extracts	Day Light	UV light
S.No	Extracts	Day Light	Short (254 nm)	Long(365 nm)
1.	n-Hexane	Dark green	Yellowish green	Fluorescent green
2.	Ethyl acetate	Blackish green	Yellowish black	Green fluorescence
3.	Ethanol	Reddish brown	Yellowish red	Black green fluorescence
4.	Aqueous	Blackish brown	Brown	Green fluorescence

Physicochemical parameters:

Physiochemical parameters are mainly used in judging the purity and quality of the powder drug. The physicochemical parameters are displayed in Table 2. Ash value of a drug gives an idea of the earthy matter or inorganic composition and other impurities present along with the drug.

The extractive values give an idea about the chemical constituents present in the drug as well as useful in the determination of exhausted and adulterated drug.

Loss on drying for the powdered drug was carried out to find out the percentage of moisture present in the drug since moisture facilitates the enzyme hydrolysis or growth of microbes which lead to deterioration. Crude fibre content so obtained can be implied to determine nutritive values.

Fluorescence analysis is an important qualitative diagnostic tool to detect the presence of chromophore in crude powdered drug under UV and day light. The fluorescence analysis of powder and extracts were given in Table 3 and 4.

TABLE – 5: Quantitative estimation of inorganic elements of the leaves of Ravenala madagascariensis Sonn.,

ELEMENT	NET COUNTS (KCps)	WEIGHT %
Calcium	33.7	20.5
Potassium	22.2	12.7
Chlorine	10.7	13
Phosphorus	2.5	3.32
Sulphur	3.9	2.83
Iron	4.1	0.813
Magnesium	0.5	0.674
Manganese	1.9	0.668

The inorganic metal analysis:

The inorganic metal analysis showed the absence of toxic metals and presence of sulphur at 2.83% could be attributed to its antidiabetic activity (Table. 5). The presence of Calcium at 20.5% and Iron at 0.813% shows its nutraceutical significance. Hence this shows the drug is absolutely safe to consume medicinally.

TABLE – 6: Preliminary photochemical screening of leaves of Ravenala madagascariensis Sonn.,

S. No	Test	Powder
1	Carbohydrates	+
2	Flavanoids	+
3	Glycosides	+
4	Alkaloids	+
5	Phenols	+
6	Tannins	+
7	Terpenoids	-
8	Saponins	+
9	Proteins	+
10	Steroids	+
11	Quinones	-
12	Furans	-

Preliminary Phytochemical Screening:

Preliminary phytochemical screening of the leaf powder showed the presence of saponins, flavanoids, tannins, phenols, glycosides, proteins and steroids (Table. 6).

CONCLUSION:

In order to select the genuine species of the drug a Pharmacognostical identity of the plant is necessary through a taxonomical and anatomical screening. The majority of the information on the identity, purity and quality of the plant material can be obtained from its macroscopy, microscopy and physico-chemical parameters. As there is no record on Pharmacognostical work on Ravenala madagascariensis Sonn., the present work is undertaken to produce some Pharmacognostical standards which can be very much useful in the identification of the drug in whole and fragmentary form. These kinds of microstructures have been recognized as tools to measure the phylogenetic relationship under light microscope to resolve the taxonomic controversies. The study also helps to establish the botanical identity for this plant that could be made use of, those who deal with the species and also in the quality assurance of the plant species.

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Received on 07.03.2010

Accepted on 13.05.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(4): July-Aug. 2010, 288-292