Keemun black tea extract contains potent fatty acid synthase inhibitors and reduces food intake and body weight of rats via oral administration

Figures & data

Figure 1 Structure of theaflavins.

Figure 2 Extracting efficiency by different solvents. Keemun black tea (1 g) was dipped in 10 ml different solvents respectively with stirring for 2 hours and the extracts were collected. The extract solution (1 μl) was added to the assay system for OA activity before initiating the reaction by FAS. The final enzyme concentration was 0.02 μM. The relative activity of FAS was measured.

Figure 3 Half-inhibition concentration (IC₅₀) on FAS of extracts with 50% ethanol from several well known Chinese teas. The final enzyme concentrations was 0.02 μM.

Table I.  Extraction efficiency of Keemun black tea under different conditions.

No.	Solvent	Time(min)	Crushed	IC50( μg tea/ml)
1	25°C50% ethanol	120	Yes	14
2	25°C50% ethanol	30	Yes	16
3	25°Cwater	30	No	150
4	75°C water	30	No	86
5	100°C water	10	No	130
6	100°C water	30	Yes	63
7	100°C water	30	No	56
8	100°C water	60	No	67
9	100°C water	720	No	68

Figure 4 Inhibition of FAS by different concentrations of Keemun BTE. The relative activity of the OA (♦), KR (•), and ER (▴) was measured. The final enzyme concentrations were 0.02 μM for OA, 0.02 μM for KR, and 0.12 μM for ER.

Figure 5 The inhibitory time course for OA (♦), KR (•) and ER (▴) of FAS in the presence of BTE. The FAS solution and BTE (in 50% ethanol) were mixed in the ratio 50:1 (v/v) in 0.1M phosphate buffer pH7.0, the final concentration of FAS and BTE being 1.9 μM and 100.6 μg dry weight/ml respectively, and aliquots were taken to assay remaining activity in these three reactions at the indicated time intervals. R.A. = relative activity to control.

Table II.  Rate constants for inactivation of FAS by Keemun BTE with protection by three substrates.

Substrate for protection	no protection	acetyl-Co	malonyl-CoA	NADPH
kobs(10^− 3min^− 1)	17.4	12.4	10.0	10.9

The concentration of FAS and the BTE was 1.9 μM and 70.7 μg dry weight/ml respectively.

Figure 6 Inhibition of FAS by different concentrations of TFs. The relative activity of the OA (♦), KR (•) was measured. The final enzyme concentration was 0.02 μM, and TFs concentrations were 0–2.6 μg/ml (3.63 μM) for OA and 0–7.7 μg/ml (10.7 μM) for KR.

Figure 7 The time course for inhibition of OA(▪) and KR(•) of FAS in the presence of TFs. The FAS and TFs solution were mixed in 0.1M phosphate buffer pH7.0, the final concentration of FAS and TFs were 1.9 μM and 26.5 μM respectively, and aliquots were taken to assay the relative activity of these two reactions at the indicated time intervals. R.A. = relative activity to control.

Table III.  Rate constants for inactivation of FAS by TFs with and without substrate protection.

Substrate for protection	no protection	acetyl-CoA	malonyl-CoA	NADPH
kobs(10^− 3min^− 1)	17.2	13	12.4	7.7

The concentration of FAS and TFs was 1.9 μM and 34.9 μM (25 μg/ml) respectively.

Figure 8 The time course for inhibition of FAS by different concentrations of TFs. The concentration of FAS in the inactivation system was 2.0 μM. (A) Observed first-order rate constants for different concentrations of TFs. (B) Plot of 1/K_obs versus 1/[TFs].

Table IV.  Inhibition patterns and parameters for EGCG, Keemun BTE and TFs.

		EGCG		BTE		TFs
	Substrate	IP	Ki( μg/ml)	IP	Ki( μg/ml)	IP	Ki( μg/ml)
OA	Acetyl CoA	C+N	17	N	13	N	0.43
	Malonyl CoA	N	21	N	28	N	1.49
	NADPH	C+N	9	C+N	4.4	C+N	0.2
KR	NADPH	C	22	C	5.6	C	2.8

IP = inhibition pattern, C = competitive, N = noncompetitive, C+N = mixed pattern of competitive with noncompetitive.

Figure 9 The effects on body weight and food intake by Keemun BTE and fenfluramine. The results are expressed as means ± s.e. of 10 SD rats. (A)The effect on the body weight over 10 days (B) The effect on the average food intake over 10 days. **P < 0.01vs. Control group, n = 10.

Table V.  Lipid index in plasma and FAS activity in liver analysis for rats.

Item	Control	Fenfluramin	Keemun BTE
Plasma Glucose (mmol/l)	5.44 ± 0.78	4.65 ± 0.43	5.13 ± 0.21
Cholesterol (mmol/l)	1.27 ± 0.13	1.63 ± 0.33	1.26 ± 0.40
Triglyceride (mmol/l)	0.29 ± 0.05	0.32 ± 0.05	0.22 ± 0.05^*
LDL (mmol/l)	0.53 ± 0.06	0.71 ± 0.2	0.56 ± 0.2
Relative FAS activity	1.0	0.73 ± 0.06	0.59 ± 0.1

* t-test P < 0.05

Figure 10 Structure of EGCG.