Morus alba L. ve Morus nigra L. üzerinde farmakognozik araştırma

Abstract

-iOl- 6. ÖZET Moraceae 60 cins vb yaklaşık 1400 ttirden oluşan bir familyadır. Morus' lar, Moraceae' nin kuzey yarım kUrenin ılımlı ve subtropikal bölgelerinde yetişen, agac ve çalılar halinde bulunan, az Üyeli bir cinsini oluştururlar. İpek böceği yetiştirilmesinde, yem olarak kullanılan yapraklarından dolayı Cin ve Japonya'da geniş alanda kültürleri yapılmaktadır. Morus alba' nın kök kabuğu (Cortex Mori) ve yaprakları halk arasında kullanılmaktadır. Eczacılık alanında bu drogun hipotansif etkisi üzerinde bazı yayınlar mevcuttur. Hipotansif etkiyi araştırmak için M. alba ve M. nigra kök kabukları sırayla n-hekzan, benzen ve metanol ile ekstre edildi. Aynı işlem metanol yerine kloroform kullanılarak tekrar yapıldı. Ekstrelerin hipotansif etkisi köpekler üzerinde denendi ve aktivite sadece M. alba kök kabuklarının metanollu ve kloroformlu ekstresinde görüldü. M.alba'nın benzenli, kloroformlu ve metanollu ekstresinden kolon kromatografisi ve P.I.T.K. ile morusin, A maddesi ve kuvanon G izole edildi. Her Uc maddenin yapı aydınlatılması UV, İR, İHNMR, 13CNMR, MS, ^C-^H- ve ^^H COSY spektrofotometrik yöntemlerle yapıldı. Yapılan I.T.K. ve miktar tayini çalışmaları M. nigra kök kabuklarında morusin ve kuvanon G'nin düşük oranda bulunduğunu gösterdi.-102- Anatomik yönden M. alba ve M. nigra kök kabukları incelenerek aralarında su farklılıklar görlildti; M. nigra da parenkima hücrelerinde görülen basit billurlara M. alba' da rastlanmadı ve sklerenkima lifi sayısı M. alba' da daha fazlaydı.

-103- 7. SUMMARY Moraceae comprise a large family of 60 genera and nearly 1400 species. Morus (mulberry) is a small genus of trees and shrubs found in temperate and subtropical regions of the Northern Hemisphere and has been widely cultivated for its leaves which serve as indenspensible food for silkworms, edible fruits and useful timber. In addition the root bark of the mulberry tree (Morus alba L. and other plants of the genus Morus) has been used as an antiphlogistic, diuretic and expectorant in Chinese herbal medicine and the crude drug is known `Shohakuhi` in Japanese and `Sang-Bai-Pi` and `Sang- P'i` in Chinese. Proir to NOMURA et.al.'s work on the the hypotensive and other phenolic constituent of this crude drug, in the pharmaceutical field a few papers had been published reporting the hypotensive effect of this drug. NOMURA et.al. were the first to isolate and identify these active substances. Prior to our work there was no report concerning the hypotensive effect of this crude drug in Iran nor Turkey, so we undertook the isolation and identification of the active substances of the mulberry root barks (Morus alba L. and M. nigra L.) collected from Iran and Turkey. The dried root barks of M.alba were finely cut and extracted successively with n-hexan, benzene and methanol. The same extraction was carried out using chloroform instead of-104- methanol. These extracts were tested for their hypotensive effects and only the methanol and chloroform extracts showed transient hypotensive effect on urethan anesthetized dogs. No marked hypotensive effect was seen in M. nigra root bark extracts. From the benzene extract using column chromatography (benzene-methanol) and preperative T.L.C. (chloroform-ether) MORUSIN was isolated (the procedure shown in Figures 6a and 6b) as yellow prisms. Morusin was recrystalized from n-hexan- ether and had the following color reactions: FeCİ3 test (green), cyanidine (orange). The structure elucidation was carried out using UV, IR, ' FABMS, EI-MS'İHNMR, 13CNMR, DEPT, 1H-13C COSY and 1H-1H COSY spectrophotometer data. Morusin gave the absorption bands for hydroxyl, conjugated carbonyl, and benzene ring in IR spectrum. The mass spectrum of morusin showed the following fragmentions: m/z 405 (M+- CH3), 203, formed from the ion at 405 by a retro Diels-Alder reaction. The NMR spectrum of morusin indicated the presence of a 2,2,-dimethylchromene ring [6 1.42, 1.55(9H, S, 16-CH3x2 and II-CH3 overlapping), 6 5.63(1H, d, J=10.3 Hz, 15-H), ö 6.57(1H, d, J=10.3 Hz, 14-H) ], since it had no signal characteristic of H-3, it was concluded that the prenyl group is located at C-3, while the signals of H-6' in the B ring was at higher field than H-6' of flavones which have no prenyl group at C-3', and T,T-dimethylallyl group [ö 1.55 (3H, s, II-CH3), 63.11 (2H,br d, J=6.8 Hz, 9-Hx2), ö 5. 11 (İH, br t, J=6.8 Hz, 10-H)]. The arrangement of substituents in-105- the B ring was assumed by a double doublet signal at ö 6.53(1H, dd, J=2.4/8.4 Hz, 5'-H), a doublet at ö 6.56(1H, d, J=2.4 Hz, 3'-H), and doublet at Ö7.23(1H, d, J=8.7 Hz, 6'-H) indicated that the B ring was substituted in the 2'- and 4*- position. The other spectra data confirm these findings. The 13CNMR spectra shows the presence of 25 carbons, 4-CH3, 1- CH2, 7-CH, 13-C. From the chloroform extract using repeated column chromatography on silica gel (chloroform, benzene-methanol) COMPOUND A was isolated. Compound A was recrystalized from n- hexan-ether to give yellow prisms and had the following color reactions: FeCİ3 test(green), cyanidine test (yellow). Compound A gave the absorption bands for hydroxyl, conjugated carbonyl, and benzene ring in IR spectrum. The *HNMR spectrum of compound A showed the presence of a 2,2-dimetilchromene ring t ö 1.46(6H, s, I6-CH3), ö 5.75(1H, d, J=10.3 Hz, 15-H), ö 6.86(1H, d, J=9.7 Hz, 14-H) ] and four aromatic protons [ö 6.14(1H, s, 6-H), ö 6.78(1H, d, J=2.4 Hz,3'-H), ö 6.66(1H, dd, J=2.4/8.79 Hz, 5'-H) Ö 7.99(1H, d, J=8.8 Hz, 6'-H)]. Although the spestrum did not show the signals of t,t- dimethylallyl group, the spectrum showed two sharp singlet signals at ö 1.32, 1.35 (each 3H, s, ll-CH3x2) and the signals of the charecteristic AMX pattern, such as ö 2. 57 (İH, dd, J=7. 4/17.1 Hz, 9-H), 6 3.46(1H, dd, J=2 Hz, 9-H), and ö 3.99(1H, dd, J=2/9.3 Hz, 10-H). From these findings it is assumed that the C5-unit is in the 3-position and this side-106- chain is cyclized with the hydroxyl group at C2*. The 13CNMR spectrum showed the presence of 25 carbons ;4-CH3, 1-CH2,7-CH and 13-C. From the methanol extract using repeated column chromatography (silica gel and polyamide) and repeated P.T.L.C. on silika gel using chloroform-methanol and n- hexan-acetone, KUWANON G was isolated as red amorphous powder and had the following color reactions: FeCl3 test (dark purple) and cyanidine test (red). Kuwanon G showed a dose-dependent trancient decrese in arterial blood pressure in anesthezed dogs. The mass spectrum of Kuwanon G showed the fragments at m/z 692 (M+, Cft0H36011), 555 (C33H3108), 420 (C25H2406), 377 (C22H1706), 147 (C9Hy06), 137 (C?H503). It is noteworthy that the fragment at m/z 420 is the same as the molecular ion of morusin. Kuwanon G gave the absorption bands of hydroxyl, conjugated carbonyl, and benzene ring in IR spectrum. *HNMR spectrum (asetone-d6) showed the following moities and groups: a 2,4-dihydroxylbenzoyl and a 2,4- dihydroxylphenyl moiety, a methil group, two methylene protons three methine protons, and an olefinic proton. The 13CNMR showed the presence of 40 carbons: 3-CH3, 2-CH2, 15- CH, and 20-C. The ^NMR and 13CNMR spectral data were-107- assigned with the results of 2D-1H,1H-CÖSY and 2D-13C, ^-COSY spectrums. From the anatomical view point the root barks of M.alba contain more schlerenchyma fibers than M. nigra. The crystalls seen in the parechyma cells near the perideme layer in M. nigra could not bee seen in M.alba. Both root barks contain large amount of starch in their wood ray and have secretory inner glands.