The effects of genetic variation and environmental factors on rhynchophylline and isorhynchophylline in <i>Uncaria macrophylla</i> Wall. from different populations in China

<div><p><i>Uncaria macrophylla</i> Wall. is an important Chinese medicinal herb. Rhynchophylline (RIN) and isorhynchophylline (IRN) are its major active compounds. We investigated the influence of genetic differentiation and environmental factors on the RIN and IRN to find the main influencing factors of their contents and lay the foundation for the following cultivation and breeding. We used inter-simple sequence repeat (ISSR) markers to investigate the genetic diversity, and high-performance liquid chromatography (HPLC) to measure the contents of RIN and IRN in 200 samples of <i>U</i>. <i>macrophylla</i> obtained from nine natural populations, and then to analyze the correlation between genetic differentiation, environmental factors of sampling sites and the contents of RIN and IRN. We found that High intra-population (80.05%) and low inter-population (19.95%) genetic diversity existed in the samples of <i>U</i>. <i>macrophylla</i>. To some extent, genetic differentiation and the contents of RIN and IRN had correlation in individual populations (such as JH, MH, XM, and ML). The RIN and IRN contents were significant negatively correlated with the precipitation in May (R_IRN = -0.771, <i>p</i> = 0.015) and June (R_RIN = -0.814, <i>p</i> = 0.008; R_IRN = -0.921, <i>p</i> = 0.000), indicating that precipitation was the main affecting factor of their contents. Interestingly, the analysis results showed that the RIN content had a significant positive correlation (r = 0.585, <i>p</i> = 0.000) with the IRN content (they are isomers); the proportion of RIN had a significant negative correlation with the sum of the two (r = –0.390, <i>p</i><0.0001), while the proportion of IRN had a significant positive correlation (r = 0.390, <i>p</i><0.0001). It meant that, with the total quantity of the two compounds increased, the proportion of RIN decreased and the proportion of IRN increased, illustrating that their conversion exist some regularity. Moreover, the content ratio of RIN and IRN was significant positively correlated with the January precipitation (r = 0.716, <i>p</i> = 0.030), implying that January may be the key period for the mutual transformation of RIN and IRN.</p></div

The PCA distribution of different populations.

<p>The PCA distribution of different populations.</p

The sampling information for 9 populations.

<p>The sampling information for 9 populations.</p

Results of correlation test based on chemical components content in 200 individuals.

<p>Results of correlation test based on chemical components content in 200 individuals.</p

Comparison between genetic dendrogram (A) and chemical dendrogram (B).

<p>Comparison between genetic dendrogram (A) and chemical dendrogram (B).</p

The contents of chemical compounds in 9 populations.

<p>The contents of chemical compounds in 9 populations.</p

Distribution of the sampling location.

<p>Distribution of the sampling location.</p

Representative CIN 1 staining patterns.

<p>Columns a-d: Four H&E stained CIN 1 specimens (a, b, d, HPV 16 positive; c, HPV 35 positive); row e-h: HPV <i>E6/E7</i> RNA CISH staining patterns; row i-l: <i>p16</i> mRNA CISH staining patterns; row m-p: p16^INK4a IHC staining patterns. The first specimen (a) may represent an early lesion displaying low-level HPV staining (e) mostly in the lower half of the lesion with occasional superficial cells showing diffusely stained nuclei (black arrows, e-h). <i>p16</i> mRNA signals were undetected. p16^INK4a staining was scored as negative. The second specimen (b) shows a productive phase HPV staining pattern as indicated by the abundant diffusely stained nuclei throughout the epithelial thickness (f). <i>p16</i> mRNA signals were barely detectable (j). p16^INK4a staining shows patchy positivity (n). Specimen c also shows a productive phase pattern of HPV expression (g). <i>p16</i> mRNA signals were detectable in the lower third of the epithelium (k) matching p16^INK4a IHC staining (o). Most CIN 1 lesions showed the staining patterns shown for specimens b, f, j, n and c, g, k, o. An exception to this staining trend was found in specimen d, which showed strong staining for HPV in the lower part of the lesion and occasional diffuse staining nuclei (h). <i>p16</i> mRNA signals were detectable in the lower epithelial layers and strong p16^INK4a staining was detected throughout the lesion; possibly, this specimen represents a CIN lesion entering into a transformative phase directly from CIN 1 morphology. All images were originally taken using a 20X objective lens. Scale bar: 50 µm.</p

Representative CIN 3 staining patterns.

<p>Columns a-d: Four H&E stained CIN 3 specimens (all HPV 16 positive); e-h: HPV <i>E6/E7</i> RNA CISH staining patterns; i-l: <i>p16</i> mRNA CISH staining patterns; m-p: p16^INK4a IHC staining patterns. All CIN 3 lesions showed staining patterns consistent with the transformative phase of HPV infection. HPV staining was detected throughout the thickness of the epithelium as strongly staining nuclear and cytoplasmic dots. Occasional diffusely staining nuclei were detected in some lesions in the outermost superficial layer; focal ‘CIN 2 patterns’ were noted along with some CIN 3 (e). <i>p16</i> mRNA staining was notable qualitatively stronger and detectable in all layers (I, j, k, l). All images were originally taken using a 20X objective lens. Scale bar: 50 µm.</p

HPV genotype distribution amongst the CIN 1, CIN 2 and CIN 3 study specimens.

<p>HPV genotype distribution amongst the CIN 1, CIN 2 and CIN 3 study specimens.</p