Ethnobotanical Leaflets 12: 461-468. 2008.

Studies on some Pharmacognostic Profiles of Bauhinia purpurea Linn.

Leaves (Caesalpinaceae)

M. Sugumaran* and  T. Vetrichelvan 

Department of Pharmaceutical chemistry, Adhiparasakthi College of Pharmacy,

 Melmaruvathur – 603 319, Tamil Nadu.

 ^*E-mail:

Issued 2 July 2008

Abstract

 The leaves of Bauhinia purpurea Linn. were studied with the aim of determining the following  pharmacognostical parameters for this species: Macroscopical characters; Leaf constants; Physico-chemical constants; Extractive values; Colour; Consistency;  Extractive values with different solvents; Micro chemical tests; Fluorescence characters of liquid extracts and leaf powder after treatment with different chemical reagents under visible and UV light at 254nm&366 nm; Measurement of cells and tissues; Bulk density  angle of repose; and, Powder microscopy. Hopefully, the determination of these characteristics will aid future investigators in their pharmacological analyses of this species.  Preliminary pytochemical studies on different extracts of the leaves were also performed.

Introduction

            Bauhinia purpurea Linn. (Caesalpinaceae ) is an ornamental plant found throughout subtropical, India, North and  South America, Nepal, Australia, Africa and United Kingdom. The plant is commonly known as Mandarai  in Tamil and khairwal in Hindi¹. The flavone glycoside, 5,6-dihydroxy-7-methoxy flavone 6-O-beta-D-xylopyranoside,  was isolated from the chloroform-soluble fraction of the ethanolic extract of bauhinia purpurea stems, and a new flavone glycoside, 6,4’ dihydroxy 3- prenyl 3,5,7,5’ tetra methoxy flavone-6-O-a-L-rhamnopyranoside, has been found in the seed of bauhinia purpurea ^{2, 3}.                                                     

            Numerous types of biological activities are attributed to bauhinia species. B. purpurea is the most important species used to treat many ailments in traditional system of medicine. (Table 1 ).^4-7 It is also reported for its antidiarrhoeal , anticancer  and  thyroid gland stimulating  properties.^8-10 Hence, country traders often subject it to adulteration/ substitution .  

Table  1.  Ethnomedical  information for bauhinia purpurea Linn.

 But, no pharmacognostical  work has been done on a drug plant of such potential until the present time. Therefore, the aim of the present investigation has been to study the important pharmacognostical characteristics of the leaves of bauhinia purpurea in both whole and powdered form.

Materials and Methods

Plant Material

The plant material was collected from  the village of Melmaruvathur (District Kancheepuram) in the month of  January 2007.  The plant was identified by local people of that village and authenticated by Dr. P. Jayaraman, Director, Plant Anatomy Research Centre (PARC), Chennai.  A herbarium specimen of the plant (MS–4) was preserved in the Department of pharmacognosy of our Institute for further reference.  The leaves were separated and dried under shade, pulverized by mechanical grinder, passed through 40 mesh sieve and stored in a closed vessel for further use. All the reagents used were of analytical grade obtained from S.D. Fine chemicals Ltd., Mumbai and Qualigens fine chemicals, Mumbai.

Methods

The macroscopical characters (size, shape, colour, odour, taste, surface, texture, venation, margin, base, and  petiole ) of the leaves were observed¹¹. Then, for powder microscopical study, the powder was stained with phloroglucinol and concentrated HCl to study the lignified cells, lignified parenchyma, trichomes, fibres, xylem vessels, mesophyll, palisade cells and stomata, etc. The powder was also stained with N/50 iodine solution to detect the presence of starch. A small portion of powder was mounted in water to identify calcium oxalate crystals¹¹. Quantitative microscopy was determined by methods prescribed by Trease and Evans¹².

            The ash values , alcohol soluble and water soluble   extractives values and Loss on drying of leaves were determined as per the Indian Pharmacopoeia methods.¹³  The crude fiber content was done by Dutch process.¹⁴ The behaviour of the powdered leaves with different chemical reagents was studied.¹⁵  The fluorescence characters of the various extracts and powdered leaf with different chemical reagents were observed  under day light and UV light (254nm&366nm), by following procedure reported by Kokoshi et al.¹⁶  Measurements of the cells/ tissues were made with the help of micrometer under a compound microscope.¹⁷ Other extractive values were determined successively starting from  petroleum ether (60-80°), chloroform, acetone, ethylacetate, methanol and distilled water by using soxhlet extraction apparatus.¹⁸  For this purpose the powder (100g ) was successively hot extracted with 300ml of above solvents for 72 h.  Before switching over to the next solvent, the powder under extraction (marc) was dried to remove the traces of earlier solvent. The dried extractives were obtained after evaporation of solvent under reduced pressure.

            The angle of repose of powder was determined by the funnel method.  The accurately weighed powder was taken in a funnel.  The height of the funnel was adjusted in such a way that the tip of the funnel just touched the apex of the heap of the powders.  The powder was allowed to flow through the funnel freely onto the surface.  The diameter of the powder cone was measured and angle of repose was calculated using the following equation, Tan q = h/r, Where h and r are the height and radius of the powder cone.¹⁹

            For the determination of bulk density, 2 g of powder, previously lightly shaken to break any agglomerates formed, was introduced into a 10 ml, measuring cylinder.  After the initial volume was observed, the cylinder was allowed to fall under its own weight onto a hard surface from the height of 2.5 cm at 2 seconds intervals.  The tapping was continued until no further change in volume was noted.  Bulk density was calculated for dried powdered drug using following formula: Bulk Density = Mass of Powder/ Bulk volume.²⁰

            Preliminary phytochemical tests of different extracts were performed by using specific reagents through standard procedures.^21-25

Results

Analysis and Discussion

            Colour: Green; Odour: Odourless; Size:8-15 cm in diameter&10-20cm long; Shape:  Shallowly cordate;   Taste: Slightly bitter;  Surface: Glabrous;  Texture: Coriaceous; Apex: marginate ; Margin: Sinuate; Venation: Parallel ; Petiole :  Size: 4-4.5 cm ; Base: Stipulate.

            The physical constant values of total ash (7.05%), acid insoluble ash (2.72%), water insoluble ash (2.98%), sulphated ash (7.91%), loss on drying (12.87%), crude fiber content (27.76%) of leaves which are specific to the plant identification. The methanol and water soluble extractive values were 35.52% and 24.4% respectively, which indicated the nature of constituents present. Quantitative microscopical study also give valuable information regarding specific leaf constants such as vein islet no  (15) , vein termination no (65), palisade ratio (78), stomatal no (3), and stomatal index (20), etc. Fluorescence studies on extracts revealed different shades of green fluorescence under UV light at 254 nm. The size of the cell elements like trichomes (66.5 -117.04 -159.6 ´  13.3 -14.3 -26.3m), starch grains (6.65-14.63-26.6m), fibers (399 -687.8-997.5 ´ 26.3-41.2-99.3) and xylem vessels (93.1 -133.4 -226.1´ 26.3 - 46.7 - 67.5 70.8m) were recorded.        

            Powder of P. dulce is pale green, fine and tasteless but with a characteristic odour.  Other features of the powder were the presence of covering trichomes, xylem vessels,  paracytic stomata, calcium oxalate crystals in the form of sheeth  and starch grains, etc. The behaviour of leaf powder (Table 2) upon treatment with different chemical reagents was also studied.

Table 2.  Behavior of powdered leaves on treatment with different chemical reagents.

Reagents                                                          Colour developed  in day light

Powder as such                                                                                      Green

1N NaOH                                                                                                 Greenish brown

Picric acid                                                                                                Yellowish green

Glacial acetic acid                                                                                   Yellowish green

1N Hcl                                                                                                      Pale yellowish green

1N HNO₃                                                                                                  Pale yellowish green

5%Iodine                                                                                                 Yellowish green

40% NaOH +few drops of 10% lead acetate                                      Yellowish green

HNO₃+ Ammonia solution                                                                    Yellow

Con H₂SO₄                                                                                               Brown

5% Fecl₃                                                                                                   Reddish brown

10% sodium hydroxide +copper sulphate                                          Green

Acetic acid +Con H₂SO₄                                                                        Green

Acetic acid +Ferric chloride +Con H₂SO₄                                           Dark blackish brown

Antimony tri chloride                                                                            Green

Ammonia solution                                                                                  Pale green

     Similarly, the fluorescence characteristic of the powdered leaf, when treated with various chemical reagents (Table 3) and  its extracts (Table 4), have also been extensively studied.

Table 3.  Fluorescence characters of the powdered leaves of    bauhinia purpurea  under UV light .

Treatments                                                                                             Colour Developed Under UV Light

                                                                                                                    Short (254nm)             Long (366 nm)

Powder as such                                                                                      Buff                                      Pale green

1N HNO₃                                                                                                  Dark green                          Pale green

5N NaOH in water                                                                                  Pale green                           Pale green

1N Hcl                                                                                                      Pale brown                          Pale green

50% HNO₃                                                                                                                                               Green                                   Green

Acetic acid                                                                                              Grey                                   Pale green

Picric acid                                                                                                Dark green                        Pale green

Fecl₃     (5%w/v aqueous solution)                                                      Black                                 Bluish black

N/20 Iodine                                                                                              Black                                 Greenish black

50% H₂SO₄                                                                                               Dark green                        Yellowish green

Ethanol                                                                                                     Green                                  Green

1N NaOH in ethanol                                                                               Reddish yellow                 Yellow

Methanol                                                                                                 Green                                  Dark green

Powder mounted with nitro cellulose                                                  Grayish white                    Greenish brown

Powder treated with NaOH in methanol, dried and

mounted with nitro cellulose                                                                Yellowish green                 Dark green

Powder treated with HCl, dried and mounted with

nitro cellulose                                                                                         Greenish yellow                Yellowish green

Powder treated with NaOH in water and mounted

with nitro cellulose                                                                                 Brown                                 Reddish brown

Powder treated with Antimony tri chloride                                        Green                                    Brown

Table 4.  Fluorescence analysis of different extracts of bauhinia purpurea.

                                                                       Colour Developed Under UV light

   Extract                                               Long ( 366 nm)                              Short ( 254 nm)

 Petroleum ether                                   Light green                                    Greenish yellow

 Chloroform                                         Greenish brown                              Dark greenish brown

 Acetone                                                Dark green                                    Light green

 Ethyl acetate                                       Blackish green                               Black

 Methanol                                            Yellowish green                             Green

 Water.                                               Greenish buff colour                        Dark green

            Fluorescence studies on extracts revealed different shades of green fluorescence under UV light at 254nm and 365nm.  After successive extraction with each solvent by using the soxhlet  apparatus , the percentage of dry extract was calculated in terms of air-dried weight and reported: Petroleum ether (6.68 %); Chloroform (1.72%); Acetone (3.24%); Ethyl acetate (2.69%); Methanol (17.93%) ; Water (18.58%), etc. The chloroform extract was minimum whereas the water extract showed the maximum extractive value, thus indicating the presence of more polar constituents in the leaf extract. Also noted were the colour  and consistency  of the extracts (Table 5 ).

Table   5 .  The colour and  consistency of  leaf extracts of bauhinia purpurea.

S.No.          Extracts                                               Colour                                  Consistency

1              Petroleum ether (60 – 80^oc) Greenish black                     Semisolid

2              Chloroform                                           Greenish black                       Semisolid

3              Acetone                                                 Greenish brown                    Semisolid

4              Ethyl acetate                                         Yellow                                    Semisolid

5              Methanol                                               Dark brown                           Semisolid

6              Water                                                     Dark brown                           Sticky

            To determine powder characters of 40- mesh size, the angle of repose and bulk density were also calculated. The drug showed an angle of repose of 44°27” and  0.4413 g/p.c. bulk density.

            The various qualitative chemical tests (Table 6) have shown the presence of phytosterols, flavonoids, fixed oils, phenolic, tannins, glycosides and saponins in huge amount; whereas, alkaloids, aromatic acids, carbohydrate, proteins and aminoacid, triterpenoids, gums, mucilage and volatile oils were totally absent in the leaf extract of this plant.

Table 6.  Preliminary phytochemical screening of various extracts of bauhinia purpurea.

Conclusion

            Macroscopic as well as microscopical studies of any phytodrug are the primary steps to establish its botanical quality control before going to other studies. As per WHO norms , botanical standards are to be proposed as a protocol for the diagnosis of the herbal drug. The above mentioned parameters are helpful for the future identification and authentification of the plant in the herbal industry and in factories. The physico-chemical standards, such as ash values, extractive values, crude fiber content and fluorescence analysis, will be useful to identify the authenticity of the drug even from the crushed or powdered plant materials.  It will serve as a standard data for the quality control of the preparations containing this plant in future.  The leaf constants can be included as microscopical standards in Indian herbal pharmacopoeia. Phytochemical study is also useful to isolate the pharmacologically active principles present in the drug.  The information obtained from the ash values and extractive values are useful during the time of collection and also during extraction process. Using these standards, the plant can be differentiated from other related species. 

Acknowledgements

The authors are grateful to the Director, NISCAIR, New Delhi for assistance in literature collection  of this plant. They are also thankful to Dr. P. Jayaraman, Director, plant anatomy research centre (PARC), chennai  for his help in the authentification  of the plant material.

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