Plant Resources, 13C-NMR Spectral Characteristic and Pharmacological Activities of Dammarane-Type Triterpenoids

Dammarane-type triterpenoids (DTT) widely distribute in various medicinal plants. They have generated a great amount of interest in the field of new drug research and development. Generally, DTT are the main bioactive ingredients abundant in Araliaceae plants, such as Panax ginseng, P. japonicas, P. notoginseng, and P. quinquefolium. Aside from Araliaceae, DTT also distribute in other families, including Betulaceae, Cucurbitaceae, Meliaceae, Rhamnaceae, and Scrophulariaceae. Until now, about 136 species belonging to 46 families have been reported to contain DTT. In this article, the genus classifications of plant sources of the botanicals that contain DTT are reviewed, with particular focus on the NMR spectral features and pharmacological activities based on literature reports, which may be benefit for the development of new drugs or food additives.

As one of the main secondary metabolites of a number of Traditional Chinese Medicines (TCM), DTT have gained more and more attention around the world owing to their remarkable biological activities [1], and display specific plant distribution. In order to complete and enrich the resource investigation of DTT, we summarize the literatures  describing this type of triterpenoids, which were extracted from various botanicals. Thus, 136 species, 79 genera, and 46 families containing DTT are summarized to reveal their plant sources.
As is known, pharmacodynamic substance research is based on structural determination, among various structural analysis methods such as ultraviolet, infrared, optical rotation, circular dichroism, nuclear magnetic resonance (NMR), and Mass spectral analysis. NMR plays an important role in structural identification. Here, the characteristics of 1 H-and 13 C-NMR spectra for DTT together with the 13 C-NMR chemical shift changes caused by various substituent groups for DTT are summarized. The work may be helpful to discriminate DTT rapidly and conveniently.
Furthermore, in pharmacological research, DTT, as well as their derivatives, showed various bioactivities such as anti-tumor, anti-inflammatory, immunostimulatory, neuronal cell proliferatory, anti-aging, anti-bacterial, anti-diabetes, and anti-osteoporosis abilities. Among the multiple DTT, ginsenoside Rg3 as the first anti-cancer monomer isolated from TCM, has been applied as a kind of auxiliary anti-cancer drug to increase efficacy and release of the chemotherapy-induced symptoms, and has been proven to be effective and safe [2,3]. Why does ginsenoside Rg3, a relatively rare DTT obtained from P. ginseng, exhibit excellent biological activity? Do other DTT perform similar ability? The explanations of their structure-activity relationships (SARs) summarized in the following might be helpful to answer these questions.
As is known, pharmacodynamic substance research is based on structural determination, among various structural analysis methods such as ultraviolet, infrared, optical rotation, circular dichroism, nuclear magnetic resonance (NMR), and Mass spectral analysis. NMR plays an important role in structural identification. Here, the characteristics of 1 H-and 13 C-NMR spectra for DTT together with the 13 C-NMR chemical shift changes caused by various substituent groups for DTT are summarized. The work may be helpful to discriminate DTT rapidly and conveniently.
Furthermore, in pharmacological research, DTT, as well as their derivatives, showed various bioactivities such as anti-tumor, anti-inflammatory, immunostimulatory, neuronal cell proliferatory, anti-aging, anti-bacterial, anti-diabetes, and anti-osteoporosis abilities. Among the multiple DTT, ginsenoside Rg 3 as the first anti-cancer monomer isolated from TCM, has been applied as a kind of auxiliary anti-cancer drug to increase efficacy and release of the chemotherapy-induced symptoms, and has been proven to be effective and safe [2,3]. Why does ginsenoside Rg 3 , a relatively rare DTT obtained from P. ginseng, exhibit excellent biological activity? Do other DTT perform similar ability? The explanations of their structure-activity relationships (SARs) summarized in the following might be helpful to answer these questions.

Plant Resources of DTT
In order to complete and enrich the resource investigation of DTT, we summarize the literatures (1965-2016) describing this type of triterpenoids, extracted from various botanicals. In Table 1, 136 species, from Araliaceae, Cucurbitaceae, Rhamnaceae, and Meliaceae families, together with 42 others are summarized .

NMR Spectral Characteristic of DTT
Meanwhile, more than 760 kinds of DTT were reported from 1965 to 2016. Summarizing the DTT NMR data, we know that the characteristics of 1 H-NMR spectra for dammarane-type sapogenin are always seven or eight singlet that belong to methyl signals in the high field δ 0.6~1.5. The chemical shift values of olefinic protons usually locate in δ 4.3~6.0, and the proton signals of oxygen carbon may appear in δ 4.0~5.5. For the 13 C-NMR spectra, the chemical shift values are usually divided in 3~4 ranges: δ 8.0~60.0 (methyl, methene, methine, and quaternary carbon; angular methyl generally located in 8~35), δ 60.0~90.0 (oxygen-methine and quaternary carbon), δ 109.0~160.0 (olefinic carbon), and 170.0~220.0 (carbonyl carbons).
Besides these NMR techniques for structure identification of natural products, application of chemical shift rules summarized from reports on similar type compounds will be useful for structure determination.
Here, the aglycone parts' NMR data of 33 representative common DTT were chosen to summarize the NMR chemical shift rules caused by varieties of substituent groups such as hydroxyl, carbonyl, olefinic bond, glycosyl, and cyclization. The work may be helpful to discriminate DTT rapidly and conveniently. As the 13 C-NMR occupy more crucial positions than their 1 H-NMR, the examples listed below were primarily elucidated by their carbon chemical shift values ( Table 2).

Carbonyl
Carbonyl always derives from the oxidation of hydroxyl. This is why carbonyl usually locates at C-3, -6, -7 and/or -12. When carbonyl appears at the C-3 position, the influence is limited to carbons right next to it as well as the C-29 methyl, all the influenced carbon signals shift downfield in different levels [20S-20-hydroxydammar-24-en-3-one (13)

Cyclization
Moreover, cyclization generally displays at C 17 -side chain. A five-membered ring with epoxy bond is usually formed between C-20 and C-24 for DTT, to maintain the consistency of deuterated solvent, here we make a δ values' comparison between betulafoliene-oxide-I (20S,24R-epoxy) (18)  Meanwhile, a five-membered lactone ring usually appears between C-21 and C-23. The effect of the lactone ring is similar to that of five-membered ring with epoxy bond, except the obvious downfield movement of C-13, the electron-withdrawing effect of the lactone group make the two olefinic

Olefinic Bond
Olefinic bond is one of the most common transformations on the side chain. In this paper, we summarize different combinations of them. The same as the other substitution forms, olefinic bond on the side chain influence not only itself but also the carbons on the D ring in different levels. In general, the substituted carbons appear characterized olefinic carbon signals on 13

Glycosyl
The hydroxyls of triterpene sapogenin are generally replaced by monosaccharide or polysaccharide, the glycosidation shifts induced by them are at the range of 8~10.

Olefinic Bond
Olefinic bond is one of the most common transformations on the side chain. In this paper, we summarize different combinations of them. The same as the other substitution forms, olefinic bond on the side chain influence not only itself but also the carbons on the D ring in different levels. In general, the substituted carbons appear characterized olefinic carbon signals on 13

Pharmacological Effects of DTT
Many herbal medicines containing DTT as major constituents have been reported for their various biological activities, including inflammation, immunodeficiency, cancer, diabetes, fungal infection, bacterial infection, osteoporosis, and central nervous system dysfunction. In this part, we summarized pharmacological activities and SARs of DTT.

Anti-Tumor activity
In TCM clinic, some herbal medicines, enriched in DTT were used as complementary and alternative agents in cancer treatment, which are helpful for preventing tumor cell metastasis, relieving side effects of radiotherapy and chemotherapy, and improving clinical cure rate, such as P. ginseng [147], P. notoginseng [148], D. binecteriferum [149], etc. Much literatures reported that DTT showed cytotoxicity in many kinds of cancer cell lines.
In vitro experiments have been carried out to analyze the cytotoxicities of DTT obtained from P. ginseng [150] (37), and (20R,24R)-dammar-20,24-epoxy-3β,6α,12β,25-tetraol (38) ( Figure 5) did not result in cytotoxicity against these human cancer cell lines. The comparison of the activities between 2 and 9, 1 and 34, and 35 and 5 indicated that the configuration at the C-20 would affect their anti-proliferative potency, and the 20S-type was stronger than the 20R-type. Moreover, their biological effects showed that PPD-type sapogenins may be a little stronger than those of PPT-type sapogenins (2 vs . 1, and 9 vs. 34). On the other hand, the results suggested that whether cyclization at the C-17 side chain (1 and 34 vs. 36-38

Pharmacological Effects of DTT
Many herbal medicines containing DTT as major constituents have been reported for their various biological activities, including inflammation, immunodeficiency, cancer, diabetes, fungal infection, bacterial infection, osteoporosis, and central nervous system dysfunction. In this part, we summarized pharmacological activities and SARs of DTT.

Anti-Tumor Activity
In TCM clinic, some herbal medicines, enriched in DTT were used as complementary and alternative agents in cancer treatment, which are helpful for preventing tumor cell metastasis, relieving side effects of radiotherapy and chemotherapy, and improving clinical cure rate, such as P. ginseng [147], P. notoginseng [148], D. binecteriferum [149], etc. Much literatures reported that DTT showed cytotoxicity in many kinds of cancer cell lines.
In vitro experiments have been carried out to analyze the cytotoxicities of DTT obtained from P. ginseng [150] 25-tetraol (37), and (20R,24R)-dammar-20,24-epoxy-3β,6α,12β,25-tetraol (38) (Figure 5) did not result in cytotoxicity against these human cancer cell lines. The comparison of the activities between 2 and 9, 1 and 34, and 35 and 5 indicated that the configuration at the C-20 would affect their anti-proliferative potency, and the 20S-type was stronger than the 20R-type. Moreover, their biological effects showed that PPD-type sapogenins may be a little stronger than those of PPT-type sapogenins (2 vs . 1, and 9 vs. 34). On the other hand, the results suggested that whether cyclization at the C-17 side chain (1 and 34 vs. 36-38), and the presence of 25-hydroxyl group (2 vs. 35, and 9 vs. 5) could play important roles in affecting the anti-proliferative potency.
Four kinds of human cancer cell lines [breast (MCF-7), lung (H838) and prostate (LNCaP (p53 wt) and PC3)] were used to determine the anti-tumor activities of ten dammarane-type terpenoids  [136], and SARs were studied. Anti-proliferative activities order of dammarane-type terpenoids on human cancer cell lines is: PPD-type > PPT-type (25-OH PPD vs. 25-OH PPT) and 25-OH PPD > PPD. Moreover, it indicated that increasing the number of sugar moieties would reduce the anti-proliferative potency. Furthermore, further anti-cancer activity evaluation with thirteen cell lines representing five types of human malignancies (glioma, pancreatic, lung, breast, and prostate) indicated that 2, 27, and 5 could inhibit the growth of all cell lines tested, and may be 5-15-fold stronger than those of 20S-ginsenoside Rg 3 (29).
Moreover, the importance of hydroxyl substitutions at C-3 and C-12 for anti-proliferative activity of DTT have been evaluated by comparing the cytotoxic activities of 20R-25-methoxyl-dammarane-3β,12β,20-triol (25R-OCH 3 PPD) (45) and its analogs substituted at the C-3 or C-3 and C-12 positions with fatty acid groups (46a-63a, and 46b-63b, Figure 5) in four different human tumor cell lines (A549, Hela, HT-29 and MCF-7) and a normal cell line (IOSE144) [152]. Consequently, compounds 45, 46a-63a, and 46b-63b showed anti-proliferative activities against all tumor cell lines with low toxicities in the normal cell line. SARs of the 45 derivatives suggested that the difference in the substituents may affect the anti-proliferative activity of the compounds. The longer the side chain of 45 is, the lower the anti-proliferative activity would be. On the other hand, the data obtained by Liu et al. indicated that C-3 and C-12 might be active sites of dammarane-type sapogenins and the hydroxyl substitutions at C-3 and C-12 would also be crucial.  [136], and SARs were studied. Anti-proliferative activities order of dammarane-type terpenoids on human cancer cell lines is: PPD-type > PPT-type (25-OH PPD vs. 25-OH PPT) and 25-OH PPD > PPD. Moreover, it indicated that increasing the number of sugar moieties would reduce the anti-proliferative potency. Furthermore, further anti-cancer activity evaluation with thirteen cell lines representing five types of human malignancies (glioma, pancreatic, lung, breast, and prostate) indicated that 2, 27, and 5 could inhibit the growth of all cell lines tested, and may be 5-15-fold stronger than those of 20S-ginsenoside Rg3 (29). It is interesting that though 25-hydroxyl group in PPD-type terpenoids has been found to play important roles in anti-proliferative potency, when it is replaced by methoxyl, the activity is still notable. For example, the IC50 values of 20S-25-methoxyl-dammarane-3β,12β,20-triol (25S-OCH3-PPD) (44) for most cell lines were in the lower μM order, which is 5-15-fold greater than 20S-PPD (2) and 10-100-fold higher than compound 29 [151].
Moreover, the importance of hydroxyl substitutions at C-3 and C-12 for anti-proliferative activity of DTT have been evaluated by comparing the cytotoxic activities of 20R-25-methoxyl-dammarane-3β,12β,20-triol (25R-OCH3 PPD) (45) and its analogs substituted at the C-3 or C-3 and C-12 positions with fatty acid groups (46a-63a, and 46b-63b, Figure 5) in four different human tumor cell lines (A549, Hela, HT-29 and MCF-7) and a normal cell line (IOSE144) [152]. Consequently, compounds 45, 46a-63a, and 46b-63b showed anti-proliferative activities against all tumor cell lines with low toxicities in the normal cell line. SARs of the 45 derivatives suggested that the difference in the substituents may affect the anti-proliferative activity of the compounds. The longer the side chain of 45 is, the lower the anti-proliferative activity would be. On the other hand, the data obtained by Liu et al. indicated that C-3 and C-12 might be active sites of dammarane-type sapogenins and the hydroxyl substitutions at C-3 and C-12 would also be crucial.  1, 2, 5, 6, 9, 27, 29, 34-45, 46a-63a, and 46b-63b. DTT have been clarified to exhibit significant inhibitory activities to breast cell lines. Bacopasides É (64) and VII (65) isolated from B. monniera [153] could remarkably inhibit human breast cancer cell line MDAMB-231 adhesion, migration and Matrigel invasion in vitro at the concentration of 50 μM. Meanwhile, both 64 and 65 showed strong inhibitory ability in mouse implanted with sarcoma S180 in vivo at 50 μmol/kg. On the other hand, both their in vitro and in vivo activities were obviously stronger than those of their homolog, bacopasaponin C (66) (IC50: 12.3, 14.3, and 34.9 μM for 64, 65,  and 66, respectively). Results revealed that the substitute positions of isobutenyl may play an important role in anti-tumor potency by comparing the activities of 65 and 66. Besides, the activity  1, 2, 5, 6, 9, 27, 29, 34-45, 46a-63a, and 46b-63b. DTT have been clarified to exhibit significant inhibitory activities to breast cell lines. Bacopasides É (64) and VII (65) isolated from B. monniera [153] could remarkably inhibit human breast cancer cell  (68) were tested. Only compound 21 was found to have significant cytotoxic activity (IC50 = 3.9 μg/mL), while 67 and 68 showed no activities, which suggested that the double bond between C-20 and C-22 of the 21,23-lactone moiety might be relatively essential for the cytotoxic activity [141]. According to the experiment carried out by Phongmaykin et al. [84], cabraleadiol (69), eichlerialactone (70), cabraleahydroxylactone (71), and cabralealactone (72) (Figure 7) found in C. penduliflorus presented weak cytotoxicity against breast cancer line with the IC50 values of 17.5, 12.5, 18.0, and 16.9 μg/mL, respectively. Among the multiple DTT summarized above, ginsenoside Rg3, one of characteristic protopanaxadiol ginsenosides of P. ginseng, has been studied the most, and has been exploited to be an effective adjuvant therapeutic agent against various cancers. Researchers have demonstrated that it could exhibit protective activities against cervical, prostate, breast, lung, gastric, colorectal, liver, and skin cancer cell lines [154].  25-triol (68) were tested. Only compound 21 was found to have significant cytotoxic activity (IC 50 = 3.9 µg/mL), while 67 and 68 showed no activities, which suggested that the double bond between C-20 and C-22 of the 21,23-lactone moiety might be relatively essential for the cytotoxic activity [141]. According to the experiment carried out by Phongmaykin et al. [84], cabraleadiol (69), eichlerialactone (70), cabraleahydroxylactone (71), and cabralealactone (72) 25-triol (68) were tested. Only compound 21 was found to have significant cytotoxic activity (IC50 = 3.9 μg/mL), while 67 and 68 showed no activities, which suggested that the double bond between C-20 and C-22 of the 21,23-lactone moiety might be relatively essential for the cytotoxic activity [141]. According to the experiment carried out by Phongmaykin et al. [84], cabraleadiol (69), eichlerialactone (70), cabraleahydroxylactone (71), and cabralealactone (72) (Figure 7) found in C. penduliflorus presented weak cytotoxicity against breast cancer line with the IC50 values of 17.5, 12.5, 18.0, and 16.9 μg/mL, respectively. Among the multiple DTT summarized above, ginsenoside Rg3, one of characteristic protopanaxadiol ginsenosides of P. ginseng, has been studied the most, and has been exploited to be an effective adjuvant therapeutic agent against various cancers. Researchers have demonstrated that it could exhibit protective activities against cervical, prostate, breast, lung, gastric, colorectal, liver, and skin cancer cell lines [154]. Among the multiple DTT summarized above, ginsenoside Rg 3 , one of characteristic protopanaxadiol ginsenosides of P. ginseng, has been studied the most, and has been exploited to be an effective adjuvant therapeutic agent against various cancers. Researchers have demonstrated that it could exhibit protective activities against cervical, prostate, breast, lung, gastric, colorectal, liver, and skin cancer cell lines [154].
The successful clinical applications of ginsenoside Rg 3 are because it can promote apoptosis, inhibit tumor angiogenesis, inhibit proliferation, invasion and metastasis of tumor cells, impact tumor gene expression signaling, reverse multi-drug resistance, and enhance immunity of patients. Currently, a clinical monomer formulation, "shenyi capsule", a capsule in combination with chemotherapy, is widely used in a variety of tumors [155].
Although ginsenoside Rg 3 shows good inhibitory effect of cancer, its poor aqueous solubility and liposolubility are not ideal for clinical applications. Recent studies hves revealed a ginsenoside Rg 3 bile salt-phosphatidylcholine-based mixed micelle system (BS-PC-MMS) that was carried out using response surface methodology based on a central composite design [156]. Thus, a proper mean for new agents like ginsenoside Rg 3 has been established to advance the studies of DTT in anti-cancer properties. On the other hand, according to the SARs mentioned above, can we revolve anticancer agent research around ginsenoside Rg 3 , and develop anti-tumor drug with high efficiency and low toxicity?

Anti-Inflammatory Activity
Inflammation is considered as the body's protective response to various chronic diseases, such as, tumor, hypertension and diabetes. The ability that DTT can inhibit inflammation has been predescribed. According to the report [157], when the MTT assay was used to evaluate the cytotoxic effects of 2α,3β,12β,20S-tetrahydroxydammar  (74) obtained from G. pentaphyllum, they showed no cytotoxicities on BEAS-2B cells in either the presence or absence of interleukin-4 (IL-4), but significantly down-regulated IL-4-induced eotaxin production in a concentration-dependent mannar, which indicated that DTT might have potential inflammatory activity, and they may be of benefit to allergic diseases. In addition, three PPT type derivations: ginsenjilinol (75), ginsenoside Rf (30), and ginsenoside Re 5 (76) isolated from the roots and rhizomes of P. ginseng have been proven to exhibit anti-inflammatory activity by inhibiting nitric oxide production by lipopolysaccharide-induced RAW 264.7 [158] (Figure 8). Although ginsenoside Rg3 shows good inhibitory effect of cancer, its poor aqueous solubility and liposolubility are not ideal for clinical applications. Recent studies hves revealed a ginsenoside Rg3 bile salt-phosphatidylcholine-based mixed micelle system (BS-PC-MMS) that was carried out using response surface methodology based on a central composite design [156]. Thus, a proper mean for new agents like ginsenoside Rg3 has been established to advance the studies of DTT in anti-cancer properties. On the other hand, according to the SARs mentioned above, can we revolve anticancer agent research around ginsenoside Rg3, and develop anti-tumor drug with high efficiency and low toxicity?

Anti-Inflammatory Activity
Inflammation is considered as the body's protective response to various chronic diseases, such as, tumor, hypertension and diabetes. The ability that DTT can inhibit inflammation has been predescribed. According to the report [157], when the MTT assay was used to evaluate the cytotoxic effects of 2α,3β,12β,20S-tetrahydroxydammar , but significantly down-regulated IL-4-induced eotaxin production in a concentration-dependent mannar, which indicated that DTT might have potential inflammatory activity, and they may be of benefit to allergic diseases. In addition, three PPT type derivations: ginsenjilinol (75), ginsenoside Rf (30), and ginsenoside Re5 (76) isolated from the roots and rhizomes of P. ginseng have been proven to exhibit anti-inflammatory activity by inhibiting nitric oxide production by lipopolysaccharide-induced RAW 264.7 [158] (Figure 8).

Immunomodulatory Activity
Immunity is a physiological function of human body. Depending on its feature of identifying "self" and "non-self" components, the antigenic material invading into the body or the cells and tumor cell damage produced by the body itself could be effectively undermined and excluded, which could keep human from being affected by a disease. 27-Demethyl-(E,E)-20(22),23-dien-3β,6α,12β-

Immunomodulatory Activity
Immunity is a physiological function of human body. Depending on its feature of identifying "self" and "non-self" components, the antigenic material invading into the body or the cells and tumor cell damage produced by the body itself could be effectively undermined and excluded, which could keep human from being affected by a disease. 27-Demethyl-(E,E)-20(22),23-dien-3β,6α,12βtrihydroxydammar-25-one (77) (Figure 9) has been excavated out from P. ginseng by the bioassay-guided assay [159]. As mentioned above, the overproduction of NO could induce not only inflammation but also the immune response. Thus, the inhibitory action of DTT on NO production has been evaluated by the study on LPS-activated mouse peritoneal macrophage. Consequently, the results implied that DTT can significantly affect cellular immunity by increasing interleukin-12 expression, Th1 response-mediated cytokine IL-2, and decreasing Th2 response-mediated cytokines IL-4 and IL-6 expression through suppressing NO production.
Generally, based on the above-mentioned studies, numerous investigations suggested that the kinds of activities of different DTT would be related to the types of aglycone and glycoside and the number of sugars linked to the dammarane skeleton. This information may be useful for evaluating the SARs of other dammarane-type sapogenins and for developing novel antineoplastic agents.
-OH in C-3 and the configuration in C-23 of the aglycone; on the other hand, the configuration of C-20 and -23 played important role to inhibitory activity of PTP1B (82 vs. 83) (Figure 9).

Conclusions
As an important secondary metabolite from numerous herbal medicines, DTT have generated a great amount of interest in the field of new drug research and development. This paper summarized plant resources, NMR spectral characteristic and pharmacological function of DTT on the basis of literatures published over the last few decades.
In the field of plant resources and NMR spectral characteristic, DTT from 46 families have been summarized. Although the planar structures of DTT have been elucidated more and more clearly by 1D and/or 2D NMR and other spectroscopes, the absolute configuration still cannot be identified comprehensively. The more precise explanation of the change of chemical shift caused by diversity substitutions should be established.
In the field of pharmacological activities, natural DTT showed various activities, including anti-cancer, anti-inflammation, immunodeficiency, anti-diabetes, and so on. Especially, SARs were deeply investigated in several kinds of tumor cell lines and animal implanted with sarcoma model, which can be utilized in future as lead compounds discovery. However, the anti-tumor mechanism and in vivo research are not enough, which restrict further application in drug development.