Junjun Zhng,Wei Zhng,Zhenhu Yin,Chngqin Li,Wenyi Kng,

a Huanghe Science and Technology College,Zhengzhou Key Laboratory of Medicinal Research,Zhengzhou,450063,China

b Institute of Chinese Materia Medica,Henan University,Kaifeng,475004,China

Keywords:

ABSTRACT

1.Introduction

Cordyceps militaris(L.er.Fr.)Link,belonging to Clavicipitaceae,Cordyceps,also known as Cordyceps,North Cordyceps,was a complex which parasitic on pupae and larvae of Lepidoptera[1].

Chemical and pharmacological researches had shown that,C.militaris contains a variety of active ingredients,including nucleosides[2],polysaccharides[3],sterols[4],fatty acids and microelement.There was strong activity in anti-oxidant[5],antifatigue[6],anti-tumor[7],anti-inflammatory[8],liver protection[9],antibacterial[10]and other aspects of aging,with important research and development value.Moreover,as a traditional Dietetic Chinese Herb,C.militaris is also the raw material of food[11]and health care alcohol[12].

Excessive blood loss was the main cause of death of war wound,accidents and surgical treatment.Therefore,hemostasis had become a hot topic in medical research.Naturally derived degradable hemostatic material,such as gelatin sponge,oxidizes cellulose and fibrin glue et al,were widely used in the treatment of skin trauma,spleen and kidney and other organ injury hemorrhages[13].However,all of them had certain deficiencies:the tissue adhesion of gelatin was poor,and its hemostasis function depends on sufficient platelets and coagulants,so their application is restricted.Fibrin glue was derived from blood and might be caused viral infections[14,15].Therefore,it was important to develop a kind of hemostatic drug with low-cost,biosecurity and effective.

It happened that this neighbour was one of those people who make mischief27 by talking too much; so, meeting the pedlar in the serai, or inn, that evening, he told him what had occurred, and added: Yes; and the young spitfire said that if beating the donkey would not do, he would beat you also, and cut your nose off for a thief! A few days later, the pedlar having moved on, two men appeared in the village inquiring who it was who had threatened to ill-treat and to murder an innocent pedlar

Hemostatic medicine of Traditional Chinese Medicine had a long history of application,and with extensive resource distribution.There were more than 300 species of traditional Chinese medicine had been proved to have compounds with coagulant activity.For instance,chimaphilin[16],isoverbascoside[17]and stavaroside D[18]all had good procoagulant activity,which were isolated from Moneses uniflora,Pedicularis striata and Stapelia variegata,respectively.Wong et al[19]found one compound from C.militaris showed strong procoagulant activity.In this paper,procoagulant activity of 70% ethanol extract of C.militaris was investigated with coagulation parameters in vitro.

2.Materials and methods

2.1.Materials and reagents

White powder with C10H13N5O3molecular formula;mp 227～228°;EI-MS m/z:251[M]+.1H-NMR(CDCl3,400 MHz)δ:1.87(1H,dd,Ha-3′),2.21(1H,dd,Hb-3′),3.50(1H,dd,Ha-5′),3.67(1H,dd,Hb-5′),4.57(1H,m,H-4′),4.84(1H,m,H-2′),4.98(1H,t,5′-OH),5.68(1H,d,J=4.4 Hz,2′-OH),5.94(1H,d,J=2.2 Hz,H-1′),7.20(2H,brs,-NH2),8.22(1H,s,H-2),8.40(1H,s,H-8).13C-NMR(CDCl3,100 MHz):151.87(C-2),148.06(C-4),119.33(C-5),156.22(C-6),139.04(C-8),90.91(C-1′),74.86(C-2′),33.95(C-3′),81.07(C-4′),62.32(C-5′).

2.2.Crude medicine

C.militaris were collected in Puyang shangpu cordyceps sinensis Co.,LTD,(Puyang,Henan,China)in April 2015,and identified by a plant scientist,Professor Changqin Li of Institute of Chinese Materia Medica,Henan University.A voucher specimen(No.20150922)was deposited in the Institute of Natural Medicine of Huanghe Science and Technology College.

Now, isn t that a fine blanket! said the old man, smoothing it over his knees. And isn t your father a kind man to be giving the old fellow a blanket like that to go away with? It cost something, it did—look at the wool of it! There ll be few blankets there the equal of this one!

2.3.Animals

Male Rex Rabbits from 2.0 to 2.5 kg were obtained from the Experimental Animal Center of Henan Province(Zhengzhou,Henan,China)(12 h light/dark cycle,25±2°C,and humidity 40%–45%)and were fed with standard rodent diet and water ad libitum for one week.

2.4.Samples extraction and isolation

Air-dried C.chinensis(1920 g)were extracted with methylene chloride at room temperature(3 times,and 3 d each time)to afford methylene chloride extract(31 g).The filtered residues were extracted with 70%(v/v)ethanol at room temperature for 3 times,and 3 d for each time.An extract was obtained after removing ethanol.The ethanol extract(260 g)was suspended in deionized water and then successively partitioned with petroleum ether,ethyl acetate,and finally with n-butanol to obtain petroleum ether extract(28 g),ethyl acetate extract(26 g)and n-butanol extract(34 g).

Ethyl acetate extract(26 g)was applied to a silica gel H mediumpressure liquid chromatography of preparation,and eluted with a stepwise-gradient of methylene chloride/methanol(100:1,100:2,100:4,100:8,100:16,100:32,0:100,in sequence)to obtain 8 fractions(Fr.1～Fr.8)by TLC analyses.Fr.1(1.05 g)was subjected to a silica gel H column chromatography and eluted with petroleum ether/ethyl acetate(from 20:1 to 1:1),then,separated on a silica gel H with petroleum ether/methylene chloride(4:1)and further chromatographed on Sephadex LH-20 to give compounds1(15.3 mg)and2(26.1 mg).Fr.3(1.64 mg)was subjected to a silica gel H column chromatography and further with Sephadex LH-20(petroleum ether/methylene chloride/methanol=9:9:2)to yield compound3(20.5 mg).Fr.4(0.86 g)was separated on silica gel H with a stepwise-gradient of petroleum ether/acetone(20:1-1:1)to obtain Fr.4.1 fraction.Fr.4.1 was separated on silica gel H with methylene chloride/acetone(15:1)and Sephadex LH-20 to give compound4(12.8 mg).Fr.5(0.70 g)was purified by silica gel H with p methylene chloride/methanol(9:1),and further with Sephadex LH-20(chloroform/methanol=1:1)to yield compounds5(3.1 mg)and6(7.2 mg).

2.5.Coagulation time assays in vitro

3.1.1.Ergosterol(1)

In the above tests,blank solvent(dimethyl sulphoxide:Tween 80:normal saline=2:8:17)was used as negative control group,while breviscapine(13.33 mg/mL)and Yunnan Baiyao(20 mg/mL)were used as positive control groups.All the samples were dissolved in blank solvent,and the concentration of compound was 5 mg/mL.

2.6.Statistical analysis

The results were expressed as arithmetic mean±standard deviation(SD).Statistical analysis was performed using SPSS19.0 software,and comparison between any two groups was evaluated using one-way analysis of variance(One-Way ANOVA).The difference between groups with P＜0.05 was regarded as statistically significant.

3.Results

3.1.Identification of the compounds

By correlating melting points and spectral data of1H-NMR,13C-NMR,and MS from the literature values,compounds1-6were identified as ergosterol(1)[21],adenosine(2)[22],cordycepin(3)[4],ergosterol peroxide(4)[23],tetracosanoic acid 2,3-dihydroxypropylester(5)[24],and mannitol(6)[25].The chemical structures of compounds1-6were shown in Fig.1.

The spectroscopic,1H and13C-NMR spectroscopic data and other physical data of the isolated compounds1-6were indicated below.

Blood samples were drawn from rabbit’s auri cular vein.After collection,the blood was placed in a centrifuge tube with 400 μL 0.109 mol/L sodium citrate to prevent blood clotting.Then serums were separated from the plasma by centrifugation of 3000 rpm at 5°for 15 min[20].Basically,serums(100 μL)was mixed with 25 μL of samples,APTT assay reagent(100 μL)was added and incubated for 5 min at 37°C,and next 0.025 mol/L CaCl2(100 μL)was added.Clotting times were recorded.PT assays,serums(100 μL)was mixed with 25 μL of samples and incubated at 37°C for 3 min.PT assay reagent(200 μL),which was hatched at 37°C for 3 min,was then added and clotting time was recorded.Meanwhile,in TT assays,serums(200 μL)was mixed with 50 μL of samples and incubated at 37°C for 3 min,and then TT assay reagent(200 μL)was added and clotting time was recorded.FIB was determined according to the manufacturer’s recommendations.

White powder with C28H44O molecular formula;mp 156～158°;EI-MS m/z:396[M]+.1H-NMR(CDCl3,400 MHz)δ:0.67(3H,s,H-18),0.80(3H,d,J=6.6 Hz,H-27),0.84(3H,d,J=6.8 Hz,H-26),0.92(3H,d,J=6.4 Hz,H-28),0.94(3H,s,H-19),1.00(3H,d,J=6.6 Hz,H-21),3.52(1H,m,H-3),5.13(1H,dd,H-22),5.20(1H,dd,H-23),5.35(1H,dd,H-8),5.60(1H,dd,H-6).13C-NMR(CDCl3,100 MHz):38.94(C-1),31.88(C-2),70.98(C-3),40.09(C-4),140.37(C-5),119.26(C-6),116.38(C-7),141.05(C-8),46.97(C-9),36.69(C-10),21.89(C-11),38.89(C-12),42.49(C-13),54.96(C-14),23.47(C-15),28.41(C-16),56.26(C-17),12.15(C-18),17.56(C-19),40.32(C-20),21.22(C-21),136.15(C-22),132.06(C-23),42.34(C-24),33.32(C-25),19.97(C-26),18.95(C-27),19.27(C-28).

3.1.6.Mannitol(6)

White powder with C10H13N5O4molecular formula;mp 236～237°;EI-MS m/z:268[M+H]+.1H-NMR(CDCl3,400 MHz)δ:3.16(1H,dd,Ha-5′),3.53(1H,dd,Hb-5′),3.77(1H,dd,H-4′),3.96(1H,m,H-3′),4.62(1H,dd,H-2′),5.28(1H,s,3′-OH),5.47(1H,dd,5′-OH),5.63(1H,d,J=6.1 Hz,2′-OH),5.83(1H,d,J=6.6 Hz,H-1′),7.19(2H,brs,-NH2),8.36(1H,s,H-2),8.47(1H,s,H-8).13CNMR(CDCl3,100 MHz):151.75(C-2),149.16(C-4),119.02(C-5),156.13(C-6),138.76(C-8),89.23(C-1′),72.00(C-2′),71.05(C-3′),85.38(C-4′),62.06(C-5′).

Nevertheless, that was what did happen, for when Queen Frivola in jest held out the fatal cup to the Princess, she took it eagerly, crying: Ah! beloved Narcissus, I come to thee! and was just raising it to her lips when the window of the great hall burst open, and the Fairy Melinette floated in upon a glowing sunset cloud, followed by the Prince himself:All the court looked on in dazzled surprise, while Potentilla, catching93 sight of her lover, dropped the cup and ran joyfully94 to meet him

Sodium chloride injection(1707182703),breviscapine injection(20161103-1),Yunnan Baiyao(2GA1604)and saline sodium citrate were obtained from Kunming Longjin pharmaceutical Co.LTD(Kunming,Yunnan,China).PT(105317),APTT(112198),TT(121181)and FIB(132120)Assay Kits were purchased from Shanghai Sun biotechnology Co.LTD(Shanghai,China).Sephadex LH-20 was purchased from Pharmacia(Burlington,MA,USA).NMR recoeded on a Bruker Avance Am-400 spectromete.

In a very cold country, far across the seas, where ice and snow cover the ground for many months in the year, there lived a little hare, who, as his father and mother were both dead, was brought up by his grandmother

Fig.1.Chemical structures of Compounds 1-6 isolated from C.militaris.

3.1.4.Ergosterol peroxide(4)

White powder with C28H44O3molecular formula;mp 178～180°;EI-MS m/z:428[M]+.1H-NMR(CDCl3,400 MHz)δ:0.80(3H,s,H-18),0.82(3H,d,J=6.7 Hz,H-26),0.84(3H,d,J=6.9 Hz,H-27),0.90(3H,s,H-19),0.95(3H,d,J=6.8 Hz,H-28),1.00(3H,d,J=6.6 Hz,H-21),3.88(1H,m,H-3),5.06(1H,dd,H-22),5.18(1H,dd,H-23),6.32(1H,d,J=8.5 Hz,H-6),6.65(1H,d,J=8.3 Hz,H-7).13C-NMR(CDCl3,100 MHz):36.04(C-1),30.25(C-2),65.86(C-3),34.92(C-4),81.73(C-5),135.05(C-6),134.78(C-7),79.16(C-8),49.92(C-9),36.60(C-10),20.79(C-11),38.87(C-12),44.19(C-13),52.06(C-14),24.47(C-15),28.11(C-16),56.13(C-17),19.15(C-18),17.23(C-19),40.25(C-20),21.20(C-21),132.25(C-22),131.36(C-23),42.54(C-24),32.32(C-25),19.77(C-26),18.95(C-27),18.23(C-28).

The results of APTT,PT,TT and FIB assays in vitro of C.chinensis were shown in Table 1.

Colorless oily liquid with C26H52O4molecular formula;mp 124～125°;EI-MS m/z:442[M]+.1H-NMR(CDCl3,400 MHz)δ:0.81(3H,m,-CH3),1.24～1.51(42 H,m,H-2′～H-23′),2.04(1H,m,H-2′),3.56(2 H,m,H-3),3.71(1H,m,H-2),4.12(1H,dd,Ha-1),4.48(1H,dd,Hb-1).13C-NMR(CDCl3,100 MHz):65.04(C-1),69.83(C-2),63.65(C-3),34.42(C-2′～C-23′),14.72(C-24′).

3.1.3.Cordycepin(3)

“That’s enough!” Mother finally laughed, when she saw that the cardboard box in the trunk was almost overflowing11. “Remember, it’s only a small crib.” So home they went, where they spread the straw carefully on a tray Mother had put on the kitchen table. The empty manger was placed gently on top, and the straw hid its one short leg.

As soon as the Wolf had uttered these words, he beat his paw against the damp ground and instantly he took the shape of the Tsar s beautiful daughter: so like to her that no one in the world could have told that he was not the Tsarevna herself. Then, bidding Helen the Beautiful wait for him outside the walls, Tsarevitch Ivan led the Gray Wolf into the Palace to Tsar Afron.

3.1.2.Adenosine(2)

What I hadn’t counted on were the people with enormous orders who chose to use the drive-thru window, or the women who felt that the coffee was much too creamy, or the men who wanted their iced coffees remade again and again until they reached a certain level of perfection. There were moments when I was exasperated4 with the human race as a whole, simply because I couldn’t seem to please anyone. There was always too much sugar, too little ice, and not enough skim milk. Nevertheless, I kept at it.

White crystal with C6H14O6molecular formula;mp 167～168°;EI-MS m/z:182[M]+.1H-NMR(CDCl3,400 MHz)δ:3.21(2H,dd,H-2,H-5),3.54(2H,dd,H-1a,H-6a),3.57(2H,d,J=8.6 Hz,H-3,H-4),3.75(2H,dd,H-1b,H-6b).13C-NMR(CDCl3,100 MHz):70.88(C-1,C-6),68.34(C-2,C-5),62.79(C-3,C-4).

3.2.Coagulation time test in vitro

3.1.5.Tetracosanoic acid 2,3-dihydroxypropylester(5)

Fig.2.Effects of compounds from C.militaris on APTT in vitro.

Fig.3.Effects of compounds from C.militaris on PT in vitro.

In the Fig.2,results indicated that compared with the blank group,ergosterol,adenosine,cordycepin,ergosterol peroxide and mannitol could significantly shorten APTT(P＜0.001).Compared with positive control group,cordycepin and ergosterol peroxide showed better effect of shorten APTT than that of Yunnan Baiyao(0.001＜P＜0.01),while the effect of adenosine and mannitol were very close.In Fig.3,compared with the blank group,ergosterol,adenosine,cordycepin,ergosterol peroxide could significantly shorten PT(P＜0.001),but the effects were worse than that of Yunnan Baiyao(P＜0.001).In the Fig.4,results indicated that adenosine,cordycepin,ergosterol peroxideandmannitol could significantly shorten TT(P＜0.001)compared with the blank group,but the effects were worse than that of Yunnan Baiyao(P＜0.001).As shown in Fig.5,compared with the blank group,ergosterol,adenosine,cordycepin,ergosterol peroxide and mannitol could significantly decrease the content of FIB(P＜0.001).Compared with Yunnan Baiyao,the effect of ergosterol peroxide was better than that of the positive control(P＜0.001),but adenosine was worse(P＜0.001).

Table 1 Effects of compounds from C.militaris on plasma coagulation parameters in vitro.

Fig.4.Effects of compounds from C.militaris on TT in vitro.

Fig.5.Effects of compounds from C.militaris on FBI in vitro.

4.Discussion

Through the chemical analysis,six compounds were isolated and purified from C.militaris:ergosterol(1),adenosine(2),cordycepin(3),ergosterol peroxide(4),tetracosanoic acid 2,3-dihydroxypropylester(5),mannitol(6).These compounds had been known to have a wide range of biological activities in studies.Lu et al[26]demonstrated that cordycepin could inhibit the proliferation of Bel-7402 cells,and induce apoptosis.Ergosterol was the characteristic alcohol of fungi,with anti-oxidant,anti-tumor and attenuation activity[27].Mannitol had anti-oxidant,antitussive and diuretic effects[28].

The coagulation process is divided into intrinsic coagulation pathways,extrinsic coagulation pathway and common coagulation pathway according to the different of activation pathway and kind of clotting factor.APTT,PT,TT,and FIB are four indicators,related to anticoagulant in clinical practice.In this study,we found that ergosterol,adenosine,cordycepin,ergosterol peroxide and mannitol from C.chinensis showed strong procoagulant activity.Compared with the blank group,adenosine,cordycepin and ergosterol peroxide could significantly shorten APTT,PT and TT and decrease the content of FIB in vitro,which indicated the procoagulant effect of them through intrinsic and extrinsic coagulation pathways,and hinder fibrin formation.Ergosterol could significantly shorten APTT and PT and decrease the content of FIB in vitro,which indicated the procoagulant effect of them through intrinsic and extrinsic coagulation pathways,and hinder fibrin formation.Mannitol could significantly shorten APTT and TT and decrease the content of FIB in vitro,which indicated the procoagulant effect of it through intrinsic coagulation pathways and hinder fibrin formation.Nevertheless,further study will be required to clarify these additional mechanisms.

A wrong order given in front of the reef- the slightest hesitation- and the boat would be lost, Then it would be all over with me and Martin too! This thought passed through Jurgen s mind one day while theywere out at sea, where his foster-father had been taken suddenlyill

Ergosterol and ergosterol peroxide had the same cyclopentanoperhydrophenanthrene steroid nucleus and side chain-5,6-dimethylhept-3-ene,The result suggested that ergosterol peroxide could prolong APTT and TT obviously led to the weak activity while the double bond at C-5 and C-8 were cleaved.Among the nucleosides possessing a side chains at N-1 and N-9,adenosine and cordycepin had the same side chain position,the structure of the side chain was different,but the procoagulant effect was no different.The data suggested that side chain structure in nucleosides was not the deciding factor for the procoagulant effect.

5.Conclusions

In the preliminary work,we had established the analysis methods of nucleoside[29]and heavy metal contents[30]of C.militaris.And on this basis we investigated the chemical constituents and the coagulation activity of C.militaris,and ergosterol,adenosine,cordycepin,ergosterol peroxide and mannitol showed strong procoagulant activity.This study showed that,C.militaris could be developed as a potential hemostatic drug.

The Fairy loved her with all her heart, for she was at once original and gentle, and she had nearly reached the age at which the gifts were generally bestowed4

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by National Key R&D Program of China(2018YFD040020)and Industry & University Research Project in Henan Province(182107000033).