BIOMEDICAL CHROMATOGRAPHY, VOL. 8, 145-147 (1994)

Studies on Standardization of Folium Strobilanthis, Radix Strobilanthis and Their Preparations

Li Ling*, Dong Tong-Yi, Li Xiu-Lu and Qiao Chuan-Zhuo School of Pharmacy, Second Military Medical University, 325 Guo He Road, Shanghai 200433, China

Determination of 4(3H)-quinazolinone, a newly found antiviral and immunopatentiating constituent was proposed as a method of evaluating the quality of two antiviral Chinese herbal drugs Daqingye (Folium Strobilanthis) and Banlangen(Radix Strobilanthis) and their preparations (‘Antipyretic and common cold granules’, ‘Banlangen granules’). An HPLC method for determining the content of 4(3H)-quinazoiinone was established with satisfactory results. The method showed good linearity (r= 0.9998, n = 5 ) in the range of 0.25-50 pg/mL of 4(3H)-quinazolinone, the average recovery was above 99% with RSD < 0.78% (n = 3).

INTRODUCTION

Dyer’s weed leaf and Isatis root (Daqingye and Banlangen in Chinese, respectively), two traditional Chinese herbal drugs, have long been used for influenza, mumps, epidemic B-type encephalitis and hepatitis. The antiviral constituents, however, have not been found until recently we isolated two novel princi- ples (Li et al . , 1993a). 4(3H)-quinazolinone(T) and 2, 4 (lH, 3H)-quinazolinedione, the former of which was found to be inhibitory on influenza virus and Coxsackie virus and to be able to promote murine splenocyte proliferation and concanavalin A (Con A) induced lym- phocyte proliferation. Therefore, it is reasonable to evaluate the quality of dyer’s weed leaf and Isatis root in terms of the content of (I). In the present study, a high pressure liquid chromatography (HPLC) method for determining the content of (I) in crude drugs and their preparations was established, and the percentages of (I) in crude drugs and preparations of different locations were assayed.

1

5 10 15 min

Figure 1. Chromatogram of standard. Injection, 10 pL; column, 250 x 4.6 mm. C18, 10 pm; mobile phase, methanol :water 4: 6 (vlv); flow rate, 1.0 mLlmin; UV detection, 230 rim; recorder sensitivity, 0.05 aufs; chart speed, 15 cmlh; room temperature. Peaks: 1, 4(3H)-quinazolinone; 2, internal standard.

* Author to whom correspondence should he addressed.

Table 1. Determination of 4(3H)-quinazolinone in the crude drugs

Sample Average content (pg/g) RSD (%) (n=41

Dukou 17.18 1.21 Chongqing 15.36 1.36 Fujian 16.52 0.97

EXPERIMENTAL

Apparatus. The chromatographic system consists of Waters series (Millipore, Milford, MA, U.S.A.), including a Model 490 programmable multiwavelength, a Model automated gradient controller, a Model 590 pump, a Model 510 pump, a Model 740 data module and a Model U6K injector equipped with a 20 pL sample loop.

Reagents and materials. Methanol, ethyl alchol absolute, tetrahydrofuran and distilled water were of analytic grade (China Industrial Standard). 4(3H)-quinazolinone was syn- thesized in this laboratory. Acetanilide (internal standard) was from Carlo Erba strumentazione (Farmitalia, Italy).

5 10 15 min

Figure 2. Chromatogram of herbal drugs. Peaks, 1, 4(3H)- quinazolinone; 2, internal standard. Other conditions as in Fig. 1.

CCC 0269-3879/94/030145-03 0 1994 by John Wiley &i Sons, Ltd.

Received 4 January I994 Accepted 24 January I994

146 LI E T A L.

~ ~~~ ~

Table 2. Determination of 4(3H)-quinazolinone in the prep- arations

Sample Average content (pgll5g) RSD ( % I ( n = 4)

Dukou 16.21 1.43 Chongqing 13.85 1.69 Fujian 14.39 1.73

5 10 15 min

Figure 3. Chromatogram of preparations. Peaks: 1, 4(3H)- quinazolinone; 2, internal standard. Other conditions as in Fig. 1.

HPLC analytic conditions. Reverse phase column, LiChrosorb RP-18(10 pm), 250 X 4.6 mm; mobile phase, methanol :water (4: 6 v/v); flow rate, 1.0 mL/min; UV detec- tion, 230 nm; recorder sensitivity; 0.050 aufs; chart speed, 15 cmlh; injection, 10 pL.

Extraction of 4(3H)-quinazolinone from the crude drugs. The crude drugs (0.5 g) were extracted with tetrahydrofuran (20 mL) using a ultrasonic extractor for 90 min. The extract was filtered and the filtrate evaporated to dryness in a water bath. The residue was dissolved in water and filtered into a 5 mL volumetric flask and added with the internal standard (87.5 pg) for HPLC analysis (extract I) .

Extraction of 4(3H)-quinazolinone from the preparations. The preparations (1.5 g) were extracted with ethyl alcohol absolute (100 mL) in a Soxhlet extractor for 5 h. The extract was concentrated to 10 mL and filtered into a 10 mL volumetric flask. The filtrate was added with the internal standard (175 pg) and used for HPLC analysis (extract 2).

Table 3. Recoveries of 4(3H)-quinazolinone in preparations Amount Amount added determined Recoven/ RSD l n = 3 )

Sample lms) Ims) % %

1 2.400 2.385 99.38 0.21 2 5.270 5.237 99.37 0.78

RESULTS

Linearity. Working solutions were prepared using ethyl alcohol absolute to give concentrations of 0.25, 12.5, 25, 37.5 and SO pg/mL and each was added with inter- nal standard (35 pg) for HPLC analysis. Regression analaysis of data obtained by running a series of work- ing solutions showed the response to be linear within the range studied (0.OS0 aufs, y = 0.0033 + 0.106Sx, r = 0.9998, where y represents peak area ratio, and x the quantity of 4(3H)-quinazolinone injected in ng). The chromatogram of the standard is shown in Fig. 1.

Sample analysis. Aliquots of 10 pL of extract 1 were analysed by HPLC. 4(3H)-quinazolinone contents in extract 1 were calculated by response factor and the results are detailed in Table 1. The chromatogram is shown in Fig. 2.

Aliquots of 10pL of extract 2 were analysed by HPLC. 4(3H)-quinazolinone contents of extract 2 were calculated by response factor and the results are detailed in Table 2. The chromatogram is shown in Fig. 3.

Reproducibility, stability and recovery. The reproducibility of the peak areas as calculated from 5 replicate analyses of standard was expressed as relative standard devi- ation (RSD) and the RSD was 1.56%. Samples were analysed on different days and the results showed that sample stability was very good within one week. The RSD was 1.48%.

The recovery value of 4(3H)-quinazolinone from the preparation was estimated using two concentration values. Extraction and analytical procedures are des- cribed above. The results obtained are summarized in Table 3 and the recoveries were in the range of 99.3- 99.4%.

DISCUSSION

‘Antipyretic and common cold granules’ and ‘Banlan- gen granules’ have been used for viral infections, and these Chinese traditional patent medicines showed a good effect. The antiviral activity and immunopaten- tiating effect of 4(3H)-quinazolinone provide a scien- tific basis for the therapeutic application of these medi- cines. Every medicine should have its own quality control standard, and the best quality control standard is the effective component of the medicine. This paper provides a good analytic method for quality control of the Chinese herbal drugs Daqingye and Banlangen as well as for their preparations (Li et af., 1993; Zhu and Zhu, 1990; Liang et al., 1990; Yu et at., 1986). We found that 4(3H)-quinazolinone possesses water solubi- lity when we carried out the extraction procedure. This confirms that the existing production procedure is effective (Li et al., 1990; Zhang et al., 1990). Besides, 4(3H)-quinazolinone also showed a good stability and these properties will make analytical work more convenient.

EVALUATION OF TWO ANTIVIRAL CHINESE HERBAL DRUGS 147

REFERENCES

Li, L., Liang, H. Q., Liao, S. X., Qiao, C. Z., Yang, G. J. and Dong,

Li, L, Qiao, C. Z., Li. X. L. and Dong, T. Y. (1993b). Acta Pharm.

Zhu, Y. G . and Zhu, X. Y. (1990). Chin. Tradit. Pat. Med. 12, 10. Liang, W. F., Bi, Y. F., Chen, T. and Lu, H. (1990). Chin. Tradit.

Herb. Drugs 21, 11. T. Y. (1993a). Acta Pharrn. Sin. 28, 238.

Sin. 28, 229. 8.

Yu, H., Jie, Q. and Li. T. Y. (1986). Chin. Tradit. Pat. Med. 1, 13. Li, Y., Teng, Z. S. and Jin, H. T. (1990). Chin. Tradit. Pat. Med. 12,

Zhang, J. J., Bo, S. Y., Zhang, Y. X. and Xu, H. L. (1990). Chin. J. Chin. Mater. Med. 15, 31.