Abstract

Several bioactive pyrrole alkaloids were identified in samples of commercial African Mango (Irvingia gabonensis) seeds which were evaluated by Li et al. for cancer chemopreventative activity[1]. Although during their follow-up screening of taxonomically authenticated African Mango seeds to confirm the results from the commercial sample, bioassays did not show the expected activity. In subsequent chemical analysis of the authentic sample, Li et al. showed that the chemopreventive pyrrole alkaloids could not be detected in the mango. Further microscopic examination of the commercial sample after treatment with Visikol showed evidence of characteristics unique to Goji Berry (Lycium barbarum).

Quality control and proper authentication of samples is critical to the manufacturing process of botanical products containing pharmaceutical or nutraceutical components, and microscopic analysis with Visikol is the easiest and fastest method to authenticate specimens and rapidly detect contaminants.  Visikol has been demonstrated to be an effective clearing agent for botanical specimens and has been shown to be a direct replacement for chloral hydrate[2], enabling researchers to clearly and quickly visually identify features that distinguish contamination or adulteration in botanical samples.

Summary

African Mango (Irvingia gabonensis) seeds have recently become available in the U.S. market as a dietary supplement and are touted for a number of positive health impacts, including: in vitro antioxidant capacity, significant effects on body weight loss, blood lipid decreases and a lowering of plasma glucose1. However, minimal data on the chemical constituents of the seeds have been reported and as a result there is potential for contamination, adulteration and/or mislabeling among samples. During the bio-guided screening of extracts of commercial African Mango for potential cancer chemopreventative agents, one novel, and three known pyrrole alkaloids, shown to enhance carcinogen detoxification by induction of the enzyme quinone reductase, were detected by Li et al (Figure 1).


However, in confirmation studies using taxonomically authenticated African Mango, no chemopreventative activity was detected. In contrast to the commercial sample, authenticated samples of African Mango seeds did not contain the expected bioactive pyrrole alkaloids. Subsequent microscopic analysis of the commercial mango sample revealed anatomical features uniquely characteristic to goji berry. Upon microscopic examination Li et al found “Irregular polygonal testa sclereids (stone cells) … with thickened and curved walls as well as distinct striations in surface view.”1 These features are characteristically of goji berry according to the American Herbal Pharmacopoeia[3], and were demonstrably absent from the taxonomically authenticated African Mango evaluated.  Further chemical analysis of authentic goji berry was able to confirm the source of these pyrrole alkaloids was indeed the goji berry.

Protocol

Grind dried samples and pass through sieve to obtain particles < 200 μm.

Fresh material can be sliced into thin sections or cleared whole.

Typically thin sections (< 1 mm) or powdered samples can be cleared directly on microscope slides by applying the sample followed by Visikol to completely cover.

Apply a coverslip, and apply gentle heat (50-60˚C) until air bubbles move to the edge of the slide.

Results

Microscopic evidence of goji berry was detected in the commercial sample, but not in authenticated sample of African Mango. The pyrrole alkaloids originally detected in the commercial sample of African Mango were subsequently detected in authenticated samples of Goji Berry by LC/MS and 1H-NMR, confirming that the presence of the bioactive pyrrole alkaloids in the commercial product was due to contamination.

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Figure 2. A) Irregular polygonal testa sclerids (stone cells) with thickened and curved walls, with distinct striations, characteristic of the seeds of goji berry (image from Li et al., 2014); B) Testa sclereids of authenticated goji berry (image from Elan Sudberg, Alkemist Pharmaceuticals, http://www.botanicalauthentication.org)

Conclusions

This surprising finding demonstrates the importance of botanical microscopy in the study of botanical products and their impact on human health. Quality control is a critical aspect for researchers and companies alike, as contamination can lead to false positives or negatives in research, mislabeled product content, and unpredictable bioactivity. With the inherent difficulty in the study of medicinally active plants, the utmost care must be taken to ensure that the product is authentic, free of contaminants, and contains the claimed level of pharmacologically relevant compounds. Visual observation under a microscope using Visikol can easily identify contamination which cannot be determined without the use of highly sophisticated and specialized techniques. The simplicity, inexpensiveness, and great utility of botanical microscopy call for its use in every lab devoted to botanical products.

 

1. Li, J., Pan, L., Naman, C. B., Deng, Y., Chai, H., Keller, W. J., & Kinghorn, A. D. (2014). Pyrrole Alkaloids with Potential Cancer Chemopreventive Activity Isolated from a Goji Berry-Contaminated Commercial Sample of African Mango.Journal of agricultural and food chemistry.

2. Villani, T. S., Koroch, A. R., & Simon, J. E. (2013). An Improved Clearing and Mounting Solution to Replace Chloral Hydrate in Microscopic Applications.Applications in Plant Sciences, 1(5).

3. American Herbal Pharmacopoeia. Lycium chinense Mill., L. barbarum L. In Botanical Pharmacognosy − Microscopic Characterization of Botanical Medicines; Upton, R., Graff, A., Jolliffe, G., Langer, R., Williamson, E., Eds.; CRC Press: Boca Raton, FL, USA, 2011; pp 468−470.