Anatomical investigation of flower of Butea monosperma Lam.

BACKGROUND: Butea frondosa Roxb. and Koen. syn. Butea monosperma Lam. (Leguminosae or Fabaceae) is a tree grows up to the height of 8 m at the age 50 years. Its flowers are being used in traditional medicine for the treatment of ulcer, inflammation, hepatic disorder, and eye diseases. AIMS: The present study was aimed at establishing the microscopic characteristics of flower B. monosperma Lam.
MATERIALS AND METHODS: Histological evaluation of flowers was done using standard procedures. Images of microscopic characters were taken at different magnifications using Nikon Labphoto 2 microscopic Unit. Perkin Elmer 5000 an atomic absorption spectrophotometer was employed for elemental analysis.
RESULTS: In the study, microscopic characters of floral parts were investigated in transverse section and the flower powder. The current study reveals the presence of pollen grains, ovary (OV), and trichomes in their flower powder. Different cell components were studied, and their sizes were measured. Elemental analysis showed the presence of Zn 52.2 μg/g and Cu 36.3 μg/g were major contents, whereas Cr, Mn, and Pd were minor contents in dried flower powder.
CONCLUSION: The current study paves the way to provide standard information related to the presence of essential elements in the flower. Microscopic characters of the flower and its quantitative measurement of cell components will help to identify the plant and also help to improvise the existing monograph of B. monosperma in the Ayurvedic pharmacopoeia.

INTRODUCTION

Butea monosperma commonly known as flame of the forest or the flame tree belongs to the subfamily “Caesalpinioideae,” of family Fabaceae or Leguminosae. It grows all over India. Various parts of this plant such as flower, bark, leaf, and seed gum are used in traditional medicine. The Ayurvedic formulations made from this plant are used to reduce the vāta and kapha among the tridoas.

Previously, many biological activities of the flower extracts with its isolated chemical constituents were studied. In this, butein, a constituent was studied and reported to have anticancer activity, prophylactic activity against inflammation and cancer; the Phytoconstituents from the extract of the flower: Butein, butrin, iso butrin, and isocoreopsin were reported to have inhibitory activity against inflammatory gene expression; its flower extract with its isolated content rutin was reported for antioxidant activity; the methanolic extract of the flowers and its isolated phytochemicals isobutrin and butrin were reported as having anti-inflammatory, anticonvulsant activities and antidiabetic, hepatoprotective effects.[1] Indian National Medicinal Plant Board has been encouraging researchers by allotting funds to develop standardization methods of selected medicinal plants of potential therapeutic significance. With this as the background, the microscopic standardization of B. monosperma is going to be a significant milestone. The current study was aimed to evaluate the histology of the much used flower of B. monosperma to improve the existing pharmacopoeia standards.

MATERIALS AND METHODS

Collection of specimens

The flowers for the present study were collected in the early morning during the summer season in March 2012 from a palāśa tree near Pattikadu in Palakkad district, Kerala. The plant material was taxonomically identified and authenticated by Prof. P. Jayaraman, Taxonomist, Plant Anatomy Research Centre, Chennai. The voucher specimen (number PARC/2010/1476) was deposited in the herbarium section of the Pharmacognosy and Phytochemistry Laboratory, Nehru College of Pharmacy, Pampady, Thiruvilwamala - 680 597, Thrissur district, Kerala state, India for future reference. 1 kg of flowers were collected, fresh flowers were used for anatomical studies and the remaining flowers were dried under shade and pulverized using a mechanical grinder and the resultant powder was stored in an airtight container to evaluate the powder microscopy. Reagents, solvents, and chemicals of analytical grade were procured from Sigma Chemical Co., St. Louis, USA and Fine Chemicals Ltd., Mumbai, Maharashtra, India.

Microscopic slide preparation

Flower of the specimen plant was fixed in FAA (formalin 5 ml + acetic acid 5 ml + 70% alcohol 90 ml). Later on the microscopic slides were prepared based on standard plant anatomy protocols.[23] The transverse sections of the flower were prepared, and staining and mounting were done according to the standard procedure.[345678] Histochemical tests were performed according to the standard methods to understand the histological characteristics of the plant.[491011] Anatomical studies were carried out with the help of references.[12] Estimation of inorganic constituents was done by standard methods.[13]

Powder microscopy

According to the Bureau of Indian standards, Mesh number 16 was used to prepare the 1 mm size powder which was then treated with 1:1 of 10% nitric acid and 10% chromic acid mixture and heated in water bath till bleaching was effected. Acid was removed from powder fragments by repeated water wash, and few drops of ammonium hydroxide were added to neutralize the powder. The pounded powder was then stained with TBO and observed under (Nikon Labphoto 2 microscopic Unit) microscope for powder characters analysis.[14]

RESULTS

Macroscopy characters of the flower of Butea monosperma

Flowers are an inflorescence with buds and open flowers. Orange to yellow colored, terminal racemes or panicles at nodes of leafless branches; rachis, pedicels, densely brown-velvety; flowers were bisexual, zygomorphic, pentamerous. Calyx, gamosepalous, appears dark, olive green to brown in color and densely velvety outside. Size 0.8–1.2 cm long, broadly campanulate, the teeth deltoid, short, the 2 upper conate in an entire or emarginate lip. Corolla about 2.0–7.0 cm long, polypetalous, comprising 2 keel petals, 2 wing petals, and one standard petal; keel petals are unequal, clothed outside with silky silvery hairs, orange or salmon colored, keel semicircular, beaked, veined; stamen, (were) (9) +1 diadelphous; anthers oblong, 2 celled uniform; style 1, long filiform, incurved, beardless; stigma 1, very small, terminal, capitate. Gynoecium, (are) monocapillary, OV sessile or shortly stalked; legumes 12–15 × 3.5–4 cm, adpressed silvery gray puberulent, apex rounded; stipe 12–15 mm. Seed reddish brown, broadly reniform or reniform-orbicular, compressed, 2.7 cm × 3.3–3.6 cm.[15161718]

Microscopy of Butea monosperma

Sepals

Thin-walled, wavy single layered upper epidermal cells (ECs) are uniseriate, multicellular trichomes and bunch of secretory canals present on lower surface, epidermis followed by three or four-layered, thin-walled, loosely arranged parenchymatous cells on both upper and lower surfaces.

Standard petal

Middle part of the standard petal was 500 μm thick. Only in epidermis, a distinct layer of small squarish thin-walled cells was observed. The outer surface has dense, nonglandular and unicellular trichomes [Figure 1.1].

Figure 1.1

T.S of standard petal through middle portion

The inner epidermis comprises small papillate ECs. The ground tissue includes outer zone of about seven layers of larger thin-walled, angular parenchyma cells, and vascular strand (VS) is found in the median part [Figure 1.1].

The marginal part of the strand petal is conical and blunt. It was measured about 80 μm thick. Both adaxial and abaxial ECs are slightly papillate [Figure 1.2]. The ground tissue consists of about six layers of compact, compressed parenchyma cells.

Figure 1.2

T.S of standard petal through marginal portion. IEP: Inner epidermis, GP: Ground parenchyma, OEP: Outer epidermis, M: Margin, VS: Vascular strand

Wing petal

The wing petal was 500 μm thick in the middle and 150 μm thick along the margin, the marginal part was thick and bluntly conical. The petal consists of thin adaxial epidermal layer of small, squarish thin-walled ECs.

The adaxial epidermis consists of prominently echinateor papillate thin-walled ECs. The ground tissue is homogeneous comprising angular, fairly wide, compact thin-walled parenchyma cells. In the median part of the petal occurs small VS [Figure 2.1].

Figure 2.1

T.S of wing petal

Keel petal

The middle part of the keel petal was curved, and it was estimated about 400 μm thick. The outer epidermis is thin, composed of small squarish cells and possess a large number of trichomes. The ground tissue consists of a median strand includes wide, angular, compact about six layers of parenchyma cells. The zone inner to the VSs is narrow and includes five or six layers of small polygonal cells [Figure 2.2].

Figure 2.2

T.S of keel petal middle portion

The marginal part of the keel petal gradually tapered into thin part toward margin. The extreme marginal part of the keel petal has outer and inner thin epidermal layers and one or two layers of ground parenchyma cells in between the epidermis. The marginal part is densely trichomatous especially on the outer side nonglandular and unicellular trichomes were seen [Figure 2.3]. The marginal part was 30 μm thick.

Figure 2.3

T.S of keel petal marginal portion. IEP: Inner epidermis, GP: Ground parenchyma, OEP: Outer epidermis, M: Margin, TR: Trichome, VS: Vascular strand

Ovary

The OV is monocarpellary with marginal placentation [Figure 3.1]. The OV wall consists of an outer thick layer of rectangular or squarish ECs. Inner to the epidermis occur a thick darkly stained tannin-containing layer of cells. The remaining portion of the OV wall consists of thin-walled, rectangular compact parenchyma cells. The ovules (OVLs) are anatropous and bitegmic. 2–5 OVLs are present inside the OV. The embryo (EM) occurs within the wide EM sac [Figure 3.2].

Figure 3.1

L.S of gynoecium

Figure 3.2

L.S of ovary with marginal OVL. EM: Embryo, OVL: Ovule, OV: Ovary, ST: Stalk, PE: Perianth, OW: Ovary wall

Powder consists of fragments of petals in surface view with long and short unicellular trichomes, numerous entire and fragments of trichomes scattered as such in the powder, the petals bear dense epidermal trichomes. The trichomes were long, thin either straight or coiled [Figure 4.1]. The trichomes were unicellular, unbranched, and uniformly thick from base to the tip. They have lignified walls and narrow lumen [Figure 4.2]. Some of the trichomes were short and thin-walled. They were unicellular and unbranched. The basal part was wide, and the terminal part was tapering. These trichomes were 400 μm to 1 mm long and 10 μm thick [Figure 5.1 and 5.2].

Figure 4.1

Outer surface of the petal with dense inter over thin trichomes. ETR: Epidermal trichome

Figure 4.2

Trichomes as seen under polarized light. ETR: Epidermal trichome

Figure 5.1

Second type of trichomes which are short and unicellular. NGTR: Nonglandular trichome

Figure 5.2

Second type of trichomes which are short and unicellular

Fragments of the petal were seen in surface view. These fragments have polygonal compact ECs of parenchyma. In some of the cells, there was a circular thick-walled cell which is the trichome bearing EC [Figure 6.1 and 6.2]. The trichome bearing cells were surrounded by about seven radially elongated rosette cells [Figure 6.1]. However, other ECs did not show any specific inclusions [Figure 6.3].

Figure 6.1

Petal fragment in surface view Darky stained cells. EC: Epidermal cells

Figure 6.2

Surface view of the petal showing a trichome bearing cell surrounded by rosette cells. RC: Rosette cell, TBC: Trichome bearing cell

Figure 6.3

A petal fragment of parenchyma cells of the wing. PA: Parenchyma

The pollen grains were triporate with three prominent thin-walled spots in the pollen wall. The surface of the pollen was smooth [Figure 7.1]. Triporate pollen grains of 40 μm diameter in the equatorial plane with a smooth surface; pollen grains appear elliptical with narrow median longitudinal slit in polar view and triangular in equatorial view [Figure 7.2]. Two celled quadrilateral anthers also were seen. Squarish ECs of the outer OV wall and inner wall made of densely arranged parenchyma cells enclosing 2–5 OVLs were observed.

Figure 7.1

Pollen grains under bright field showing polar view and equatorial views. PV: Polar view, EV: Equatorial views

Figure 7.2

Pollen grains as seen under dark field microscope showing equatorial and polar views. P: Pollen grains, PV: Polar view, EV: Equatorial views

Elemental analysis

Elemental analysis showed the presence of Zn 52.2 μg/g and Cu 36.3 μg/g were major contents, whereas Cr, Mn, and Pd were minor contents [Table 1].

Table 1

Elemental analysis of flower of Butea monosperma

DISCUSSION

The detailed histological study of B. monosperma has been carried out in the present study, which helps to identify this species without any confusion. The most convenient and cost effective method of identification of a medicinal herb would be the use of microscopic characteristics, it has been the foundation of conventional pharmacognosy and remains a fundamental module of the contemporary monograph.[19]

Flower is a unique character of plant identification, the petal arrangement in this plant plays a major role in identifying this plant. Even though Ayurvedic pharmacopoeia has included a monograph of this flower, an elaborate microscopic study of the flower is crucial as flower has been used in traditional medicine.

Powder microscopy also plays an important role in pharmacognostical evaluation and sometimes may be an identifying parameter of herbal drugs. According to quantitative microscopy, the middle part of the standard petal was 500 μm thick; the marginal part of the strand petal was 80 μm thick; the wing petal was 500 μm thick in the middle and 150 μm thick along the margin. The middle part of the keel petal was curved, and it was 400 μm thick. The petal has long epidermal trichomes of 400 μm to 1 mm length and 10 μm thickness. The pollen grains were triporate with 40 μm diameter in the equatorial plane. The OV is monocarpellary, and the OVLs were anatropous and bitegmic. Stamens was diadelphous; pollen grains are spheroidal; about 28 μm long and 30 μm broad, pore oval to elongate, 8–12.5 μm exine wall surface foveolate.[16] These are evaluated in the first time in our studies which would stand as unique quality control parameters towards identification of flowers of this plant.

Interestingly, during the present study, mineral elements in the dried flower powder were found produce significant therapeutic effects in the human body. Minerals also responsible of imparting color to the natural products. Chromium imparts orange color, whereas Copper imparts red color and Manganese imparts orange yellow color.[20] The present study has found the presence of mineral elements in the dried flower powder. Various studies have shown minerals as having therapeutic effects in the humans. Copper deficiency results in chronic or repeated diarrhea and low resistance to infection and anemia. Manganese is needed for hemoglobin development, reproduction, and skeleton growth in humans and to cure skin illnesses, scabies, piles, and rheumatism. Zinc is composed of many metalloenzymes and also is membrane stabilizer and stimulator of the immune response. Its deficiency leads to loss of appetite and weakens immune function and hair loss, delayed sexual maturation, impotence and hypogonadism in males, and eye and skin lesions. Chromium assists carbohydrate metabolism and supports transmission in the neuromuscular system its deficiency causes diabetes in humans.[21]

CONCLUSION

The pharmacognostic evaluation of the transverse section and powder form of B. monosperma flowers was conducted, and the presences of important elements in the powder were established. The macro and microscopic profile of the flower can be used as standard data for identification of B. monosperma species from its adulterants and its substitutes.