control com pound + isolated compound 4: inactive

Figure 5.5 IC 50 Curves of control compounds and the control compounds with added natural products

CHA D isp la cem en t (A d e n o s in e 1 R ece p to r) 100 ■ CHA * CHA* Compound 1 7 5 0 g [ D is p la c e r ] nM

G libenclam ide D isp la ce m e n t (K + -c h a n n e l R e c e p to r )

100

■ G lib en clam ide

g

2

- 2

[D is p la c e r ] nM

G lib en clam ide D isp la cem e n t (K + -c h a n n e l R e c e p to r )

100

■ G lib en clam ide

*• Glib + C o m p o u n d 1 9

I

I

G li b e n cl am id e D i s p l a c e m e n t ( K + - c h a n n e i R e c e p t o r ) 100 ■ G lib en cla m id e Glib + C om pou n d 2 0 m 50 § - 2 0 1 2 3 [D is p la c e r ] nM

N itr e n d ip in e D isp la cem en t (C a + + -c h a n n e l r e c e p t o r ) 100 ■ N itr e n d ip in e A N itre.+ C om p ou n d 2 0 œ 50 -2 [D is p la c e r ] nM

CHA D isp la cem en t ( A d e n o s in e 1 R e c e p to r ) 100 - CHA A CHA+Compound 2 0

I

I

CHA D i s p la c e m e n t ( A d e n o s i n e 1 R e c e p t o r ) 100 • CHA m CHA+Compound 21 50 g [D is p la c e r] nM

DMA D isplacem ent (B -a d re n o c e p to r) 100 propranolol pro.*-Compound 25 m 50 § [D is p la c e r] nM

DMA D isplacem ent (0 -a d r e n o c e p to r) 100 propranolol prOi+Compound 26 50 § [ D i s p l a c e r ] nM

5.5 Discussion

1. The constituents isolated from Clerodendrum mandarinorum are all non-nitrogen containing. Their structures differ from the nitrogen-containing synthetic compounds used clinically for the treatment of the CNS disorders. Compounds 16, 21, 25 were mildly active against K^-channel, adenosine 1 and muscarinic receptors (IC5 0 3.0-7.5

pM, Table 5.5, p. 150). In interaction experiments, 17,19, 20 had marked influence on ion channel receptors (Ca^^, K^-channel receptors), reducing the binding sites of the receptors (Figure 5.5, p. 153). These findings suggested that the principles isolated from this plant may exert their effects either by the activities at high concentration or by influence on the binding of other compounds (endogenous or exogenous) to the receptors. The further function assays are needed in order to understand the mechanism of action of these natural products.

2. The root bark extract of C. mandarinorum showed a strong binding to adenosine 1 receptor. Following the separation of active fractions, compound 21, a flavonoid glucoside, was found to be the active constituent (IC5 0 3.0 pM, Table 5.5, p. 150). It

is of interest that all known adenosine agonists are closely related to adenosine itself, and very few modifications to its basic structure are allowed. (Galen, et al, 1992). For the adenosine antagonists, they are generally planar, aromatic (or having a high n- electron density), nitrogen-containing heterocycles. The most potent receptor sensitives are 6:5-fused bicyclic or 6:6:5-fused tricyclic heterocycles. The naturally occurring benzo[b]furan is an only exception of this general rule which contains an O- rather than a N- 6:5-fused heterocycle, yet maintains considerable potency (Ki 17nM) in binding to the adenosine 1 receptor (Galen et al, 1992). The structure of compound 21 has some similarity to benzo[b]furan (Figure 5.6). It has not been shown previously that flavonoids are able to bind with adenosine 1 receptor and compound 2 1 is first

one to be observed with this activity. The structure of compound 21 is specific to A l receptor since none of the other eight flavonoids tested were active, even for sorbifolin-4’-glucose whose structure is very close to compound 21. In interaction experiments, compound 21 showed an effect on the binding of CHA (an agonist of A l receptor) to the receptor, and this fact may be related to its activity at A l receptor at high concentration.

OMe .OGlc OMe RO' OH O compound 21 R=CH3 sorbifolin-4'-Glucose R=H benzo[b]furan

Figure 5.6 Structures of benzo[b]furan, compound 21 and sorbifolin-4-glucose

3. The flavonoids are one of the largest groups of natural products. Several flavonoids are obtained in the present study and they are also present in many species of Chinese herbs. The diverse pharmacological activities of flavonoids have been described (Havsteen, 1983). Activities include their effects on the synthesis of prostaglandins, on various enzyme systems and effects as anti allergic, anti-inflammatory, and anti­ asthmatics. In the present study, two flavonoids (compound 20, 21) have been tested for their interaction effect on the control compounds, and both of them showed the influence on specific receptors, such as histamine 1, adenosine 1, Ca^^-channel and K^- channel receptors. The synergistic effects of flavonoids have been found in a number of previous studies. For instance, it was reported that some flavonoids (eg. quercetin) were able to potentiate or to inhibit the release of histamine by rat peritoneal mast cell induced by a compound (called compound 48/80) according to the concentration used. (Amellal, et al, 1985). It was also found that the antimalarial activity of artemisinin (qing haosu) was markedly enhanced by the presence of methylated flavones such as artemetin and casticin from Artemsia annua L. (Yang, et al, 1988). It is not inconceivable that flavonoids may act through a variety of different mechanisms (eg. on membrane ATPase, or on prostaglandins synthetase). Low activity and large doses needed are common disadvantages of the flavonoids, on the other hand, they have generally lower toxicity and offer a wide variety of chemical structures. The rational utilization of this group of compounds, especially for their synergistic effects, may be beneficial to new drug development. Moreover, the investigation into the functions of

flavonoids at the molecular level will further increase our understanding of their actions in herbal medicines.

4. Five oligosaccharides have been isolated from this plant and they are the major group of compounds present in the highly polar fractions. Meanwhile, the saccharides are also the main constituents of the herb decoction. Compound 25 showed weak activity against the muscarinic receptor (IC5 0 5.5 pM, Table 5.5, p. 153) and

compounds 25 and 26 exhibited interactions on the control compounds binding to histamine 1, 5HT1A and a2-adrenoceptor(Table 5.6, p. 152). Glucose is the basic unit of these compounds which is almost the sole substance for cerebral oxidative metabolism (Bachelard, 1978; Sokoloff, 1977). Therefore, further investigation of the functions of saccharides may help understand the action mechanism of the plant decoction.

Chapter 6