Determination of chlorophylls and their derivatives in Gynostemma pentaphyllum Makino by liquid chromatography–mass spectrometry

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Abstract

The objectives of this study were to develop a high performance liquid chromatography–mass spectrometry (HPLC–MS) method for determination of chlorophylls and their derivatives in Gynostemma pentaphyllum Makino, a traditional Chinese herb possessing vital biological activities. Chlorophylls were extracted with a quaternary solvent system of hexane–acetone–ethanol–toluene (10:7:6:7, v/v/v/v), followed by separation of a total of 15 chlorophylls and their derivatives within 32 min using a gradient mobile phase of acetone, acetonitrile and methanol and a HyPURITY C18 column, with detection at 660 nm and flow rate at 1 mL/min. Identification was carried out on the basis of retention behavior, absorption spectra and mass spectra using atmospheric pressure chemical ionization (APCI) in positive ion mode for detection. Of the 15 analytes, chlorophyll a, chlorophyll b, pheophytin a and pheophytin b were quantified by using standard calibration curves, with the other 11 being quantified with an internal standard Fast Green FCF. Chlorophyll extracts in G. pentaphyllum were found to contain pheophytin a (2508.3 μg/g), pheophytin a′ (111.2 μg/g), chlorophyll a (113.8 μg/g), chlorophyll a′ (11.0 μg/g), hydroxypheophytin a (88.6 μg/g), hydroxypheophytin a′ (66.5 μg/g), pyropheophytin a (76.0 μg/g), hydroxychlorophyll a (23.8 μg/g), pheophytin b (319.6 μg/g), pheophytin b′ (13.2 μg/g), chlorophyll b (287.9 μg/g), chlorophyll b′ (11.1 μg/g), hydroxychlorophyll b (15.0 μg/g), hydroxypheophytin b (11.2 μg/g) and hydroxypheophytin b′ (8.5 μg/g).

Introduction

Chlorophyll, a photosynthetic pigment that is widely distributed in nature, possesses a basic skeleton structure of porphyrine with a magnesium ion in the center and a long phytol group in the tail [1]. The major chlorophylls in plants include chlorophyll a and chlorophyll b, which are usually present at a ratio of 3:1 [2]. In addition to chlorophyll a and chlorophyll b, there are several chlorophyll derivatives such as pheophytin a, pheophytin b, pyropheophytin a, pyropheophytin b, pheophorbide a, pheophorbide b, chlorophyllide a and chlorophyllide b present in plants [1]. All these derivatives could be formed through heat or acidic treatment or enzymatic degradation [2]. For instance, pheophytin a and pheophytin b could be formed from chlorophyll a and chlorophyll b during cooking of green vegetables, respectively, whereas chlorophyllide a and chlorophyllide b could be formed from chlorophyll a and chlorophyll b in the presence of chlorophyllase [3], [4].

Chlorophylls and their derivatives have been extensively studied for their biological activities. Lanfer-Marquez et al. [5] reported that pheophorbide b and pheophytin b were the strongest natural antioxidant compounds, revealing the importance of the aldehyde group for antioxidant activity. In another study dealing with the effect of chlorophyllin on radiation-induced immunosuppression and modulation of immune responses, Sharma et al. [6] indicated that chlorophyllin could inhibit the in vitro lymphocyte proliferation induced by concanvalin A in a dose-dependent manner, and the expression of antiapoptoic genes bcl-2 and bcl-xL was up-regulated in spleen cells. In addition, the antigenotoxic activity against the DNA damage by chlorophyll a, chlorophyll b and chlorophyllin, as well as the inhibition of skin tumor formation in ICR mouse by pheophorbide a have been demonstrated [7], [8]. Nevertheless, several studies have shown that both chlorophyll and chlorophyllin may result in genotoxic and carcinogenic effects [9], [10]. Therefore, the impact of chlorophylls and their derivatives on human health cannot be ignored.

Gynostemma pentaphyllum (Thunb.) Makino, a traditional Chinese herb that is frequently used in the treatment of chronic disease by oriental people, has been shown to possess several vital biological activities like anti-cancer and anti-inflammation as well as liver protection [11], [12]. The presence of functional components in G. pentaphyllum such as flavonoids, saponins, carotenoids and chlorophylls are believed to be responsible for this beneficial effect. However, the amount and variety of chlorophylls in G. pentaphyllum still remain unknown. The objectives of this study were to develop a high performance liquid chromatography–mass spectrometry (HPLC–MS) method for determination of chlorophylls and their derivatives in G. pentaphyllum.

Section snippets

Materials

Both chlorophyll a and chlorophyll b standards were purchased from Sigma (St. Louis, MO, USA). Internal standard Fast Green FCF was procured from Fluka (Buchs, Switzerland). Pheophytin a and pheophytin b were prepared from chlorophyll a and chlorophyll b, respectively, using a method as described by Teng and Chen [13]. Pyropheophytin a was prepared from pheophytin a based on a procedure by Pennington et al. [14]. Briefly, 1-mL of pheophytin a in acetone was evaporated to dryness under nitrogen,

HPLC separation of chlorophylls and their derivatives

In the beginning several solvent systems in isocratic or gradient mode reported in the literature were used for separation of chlorophylls and their derivatives in G. pentaphyllum [17], [18], [19], [20], [21]. However, these methods fail to resolve chlorophylls and their derivatives in G. pentaphyllum, probably caused by the presence of different varieties of chlorophyll derivatives. Thus a new solvent system has to be developed. By calculating the polarity index (solvent strength) of each

Conclusion

In conclusion, a gradient ternary solvent system of acetone–acetonitrile–methanol was developed to separate chlorophylls and their derivatives in G. pentaphyllum by HPLC, and a total of 15 pigments including pheophytin a, pheophytin a′, chlorophyll a, chlorophyll a′, hydroxypheophytin a, hydroxypheophytin a′, pyropheophytin a, hydroxychlorophyll a, pheophytin b, pheophytin b′, chlorophyll b, chlorophyll b′, hydroxychlorophyll b, hydroxypheophytin b and hydroxypheophytin b′ were resolved within

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